Design of a cyclopropyl quinone methide reductive alkylating agent. 2

Article abstract of DOI:10.1021/jo0602423

A cyclopropyl quinone methide is formed by elimination of a leaving group from an appropriately functionalized hydroquinone. The presence of a carbon spacer results in the formation of a fused ring rather than the classic methide species. Discussed herein

Full text of DOI:10.1021/jo0602423

Design of a Cyclopropyl Quinone Methide Reductive
Alkylating Agent. 2
Omar Khdour, Anlong Ouyang, and Edward B. Skibo*
Department of Chemistry and Biochemistry, Arizona State UniVersity, Tempe, Arizona 85287-1604
eskibo@asu.edu
ReceiVed February 6, 2006
A cyclopropyl quinone methide is formed by elimination of a leaving group from an appropriately
functionalized hydroquinone. The presence of a carbon spacer results in the formation of a fused ring
rather than the classic methide species. Discussed herein is cyclopropyl quinone methide formation from
a pyrido[1,2-a]indole ring system. Both nucleophilic and electrophilic attack on the fused cyclopropane
ring results in pyrido[1,2-a]indole and azepino[1,2-a]indole products. The stereoelectronic effect plays
less a role in the relatively flexible pyrido[1,2-a]indole system compared to its role in the pyrrolo[1,2-a]-
indole system. A 13C label on the fused cyclopropane ring permitted the rapid identification of complex
rearrangement products observed in this study.
Introduction
and the A-ring of CC-1065.14 While the mitosene hydroquinone
affords the quinone methide by elimination of a leaving group,
the present hydroquinone systems can eliminate the leaving
group only by formation of a fused cyclopropane ring.
Quinone methide chemistry is of general interest since many
naturally occurring and synthetic quinones can form this reactive
species upon two-electron reduction and leaving group elimi-
nation.1-9 Previously, we designed a system that could form a
cyclopropyl quinone methide from a pyrrolo[1,2-a]-fused system
upon reduction of the quinone.10 The inspiration for the cyclo-
propyl quinone methide design came from the mitosenes11-13
Presented herein are the synthesis of 1, and the study of the
fate of its cyclopropyl quinone methide 2, Scheme 1. A 13C
label at the starred center of 1 permitted the rapid identification
of products resulting from the nucleophile-mediated opening
of the cyclopropane ring to afford ring retention and ring
expansion products, 3 and 4, respectively, in the inset of Scheme
1. This study represents a continuation of our use of 13C labeling
to elucidate methide chemistry.10,15,16 The stereoelectronic effect,
along with steric effects, controls the nucleophilic attack on the
fused cyclopropane ring resulting in different ring opening paths
for pyrido[1,2-a]-fused cyclopropyl quinone methide (2) com-
pared to the previously reported pyrrolo[1,2-a]-fused analogue
5,10 Scheme 2. The more flexible pyrido[1,2-a] ring of 2 permits
both nucleophilic attacks shown in the inset of Scheme 1
resulting in formation of both pyrido[1,2-a]indole 3 and azepino-
[1,2-a]indole 4, with the latter favored by large nucleophiles.
In contrast, the pyrrolo[1,2-a]-fused system 5 affords only 6
* To whom correspondence should be addressed. Phone: 480-965-3581.
Fax: 480-965-2747.
(1) Moore, H. W. Science (Washington, D. C.) 1977, 197, 527-532.
(2) Moore, H. W.; Czerniak, R. Med. Res. ReV. 1981, 1, 249-280.
(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.
(5) Lee, C.-H.; Skibo, E. B. Biochemistry 1987, 26, 7355-7362.
(6) Skibo, E. B. J. Org. Chem. 1992, 57, 5874-5878.
(7) Zeng, Q. P.; Rokita, S. E. J. Org. Chem. 1996, 61, 9080-9081.
(8) Rokita, S. E.; Yang, J. H.; Pande, P.; Greenberg, W. A. J. Org. Chem.
1997, 62, 3010-3012.
(9) Maliepaard, M.; deMol, N. J.; Tomasz, M.; Gargiulo, D.; Janssen,
L. H. M.; vanDuynhoven, J. P. M.; vanVelzen, E. J. J.; Verboom, W.;
Reinhoudt, D. N. Biochemistry 1997, 36, 9211-9220.
(10) Ouyang, A.; Skibo, E. B. J. Org. Chem. 1998, 63, 1893-1900.
(11) Remers, W. A. In The Chemistry of Antitumor Antibiotics; John
Wiley & Sons Inc.: New York, 1979; Vol. 1, p 221-276.
(12) Franck, R. W.; Tomasz, M. In The Chemistry of Antitumor Agents;
Wilman, D. E., Ed.; Blackie & Sons, Ltd.: Glasgow, Scotland, 1990; p
379-394.
(14) Boger, D. L.; Johnson, D. S. Proc. Nat. Acad. Sci. U.S.A. 1995, 92,
3642-3649.
(15) Ouyang, A.; Skibo, E. B. Biochemistry 2000, 39, 5817-5830.
(16) Skibo, E. B.; Xing, C.; Groy, T. Bioorg. Med. Chem. 2001, 9, 2445-
2459.
(13) Boruah, R. C.; Skibo, E. B. J. Org. Chem. 1995, 60, 2232-2243.
10.1021/jo0602423 CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/11/2006
J. Org. Chem. 2006, 71, 5855-5863
5855

Khdour et al.
SCHEME 1
SCHEME 3
SCHEME 4
SCHEME 2
upon nucleophilic attack regardless of the size of the nucleo-
phile.10 The pyrido-fused product 6 eliminates the nucleophile
to afford, upon air oxidation, the aromatized product 7. In con-
trast, the nucleophile trapping products of cyclopropyl quinone
methide 2 do not eliminate readily because products are not
aromatic.
was then methylated, followed by reductive cyclization to the
indole 9. The presence of the 3-methyl group prevented
substitution reactions at this position of the indole ring during
the synthesis of 1. The annulation of the pyrido ring to afford
the pyrido[1,2-a]indole 10 was accomplished by condensation
of 9 with γ-butyrolactone followed by dehydrative cyclization
using hot polyphosphoric acid. This annulation procedure has
been employed for the preparation of other pyrido[1,2-a]indole
analogues.19
Results and Discussion
Synthesis. The synthesis of 1 was accomplished in 10 steps
from commercially available 2-methyl-4-nitroanisole as outlined
in Schemes 3 and 4. Introduction of the acetonitrile moiety into
this starting material was carried out through “vicarious nu-
cleophilic substitution” to afford 8.17,18 The acetonitrile moiety
Nitration of 10 to afford 11 was carried out in dichloro-
methane with 67% nitric acid. Replacement of the ketone group
of 11 with a cyano group to afford 12 was carried out by
(17) Makosza, M.; Danikiewicz, W.; Wojciechowski, K. Liebigs Ann.
Chem. 1988, 203-208.
(18) Marino, J. P.; Hurt, C. R. Synth. Commun. 1994, 24, 839-848.
(19) Hosseini, S. H.; Entezami, A. A. J. Appl. Polym. Sci. 2003, 90,
63-71.
5856 J. Org. Chem., Vol. 71, No. 16, 2006

Cyclopropyl Quinone Methide Alkylating Agent
SCHEME 5
SCHEME 6
Cyclopropyl Quinone Methide Chemistry. The product
studies described below provided indirect evidence for the
formation of the cyclopropyl quinone methide species 2 shown
in Scheme 1. Product studies revealed that 2 has three possible
fates: proton (electrophile) trapping resulting in alkane forma-
tion, nucleophile trapping, and dimerization (which is a com-
bination of electrophile and nucleophile trapping). Some of these
products are converted to other products by tautomerization
processes. Unlike the cyclopropyl quinone methide 5,10 shown
in Scheme 2, the stereoelectronic effect predicts no preferred
path for cyclopropane ring opening because of ring flexibility.
Reduction of 1 and incubation in methanol followed by
aerobic workup afforded a complex mixture of products that
were initially analyzed by 13C NMR, Figure 1. This spectrum
clearly shows the formation of four types of products based on
the chemical shifts of the label: oxygen-substituted 13C (74
ppm), allylic or benzylic 13C (45 ppm), 13C with oxygen on a
neighboring carbon (29 ppm), and alkyl 13C (21 ppm). Isolation
and identification studies confirmed the presence of each of these
types of products, Scheme 7. Nucleophilic attack on the
cyclopropyl quinone methide 2 affords pyrido[1,2-a]indole 21
and azepino[1,2-a]indole 22 upon oxidative workup as illustrated
in Scheme 7. Initially, nucleophilic attack on the cyclopropane
ring of 2 affords the hydroquinone derivatives that are oxidized
to the quinones upon aerobic workup. Inspection of the
minimized molecular model of 2 in Figure 2 reveals that pyrido-
[1,2-a]indole and azepino[1,2-a]indole formation upon nucleo-
philic attack are both favored by the stereoelectronic effect.
Ideally, the breaking bond is 45° to 90° to the π system so that
there is maximal overlap with the developing p orbital. For
example, nucleophilic attack of the CC-1065 A ring at the least
substituted carbon of the cyclopropane ring, and subsequent
bond breaking, results in good overlap (45° out of the π plane)
of the developing p orbital with the π system.23 In contrast,
attack at the more substituted carbon would result in p orbital
development orthogonal to the π system. Similarly, the stereo-
electronic effect correctly predicted that cyclopropyl quinone 5
would undergo nucleophilic attack only at the more substituted
carbon.10 In contrast to 5 and the CC-1065 A ring, both
nucleophilic attack paths on 2 will result in good overlap
between the developing p orbital and the π system. The greater
flexibility of the tetrahydropyrido ring of 2, compared to that
treatment with tosylmethyl isocyanide anion.20,21 The resulting
nitrile 12 was then exchanged with Na13CN in DMSO at 80 °C
to afford the 13C enriched nitrile analogue 12* in a 50% yield.
Dry HCl was added to pyrido[1,2-a]indole nitrile 12 in methanol
solution resulting in formation of the imino ester, which was
treated with 3N HCl for 24 h to afford 13. The conversion of
13 to hydroxymethyl pyrido[1,2-a]indole 14 as illustrated in
Scheme 4 was uneventful. However, the conversion of 14 to
15 by catalytic reduction/Fremy oxidation at pH ) 3 occurred
in low yield with noteworthy side reactions discussed below.
Finally, the hydroxymethyl indolequinone 15 was treated with
methanesulfonyl chloride/pyridine to give methanesulfonoxy-
methyl indolequione 1.
Fremy oxidation of reduced 14 (nitro to amino) afforded a
variety of products depending on the pH of the reaction, Scheme
5. All reaction mixtures afford varying quantities of the diol
16, a reaction not seen with the pyrrolo[1,2-a] fused analogues.10
It is proposed that indole quinone 15 readily tautomerizes to
the quinone methide species that either traps water or loses a
proton to afford 16 and 17, respectively (Scheme 6). The ease
of tautomerization of the pyrido[1,2-a] fused analogues is
attributed to less strain of the methide when present in a six-
membered ring. The high yield preparation of 16 was carried
out by Fremy oxidation to the iminoquinone at pH 722 to afford
imino-16 followed by acid-catalyzed imine hydrolysis.
(20) Oldenziel, O. H.; van Leusen, A. M. Synth. Commun. 1972, 2 (5),
281-283.
(21) Oldenziel, O. H.; van Leusen, D.; van Leusen, A. M. J. Org. Chem.
1977, 42, 3114-3118.
(22) Islam, I.; Skibo, E. B. J. Org. Chem. 1990, 55, 3195-3205.
(23) Boger, D. L.; Johnson, D. S. Angew. Chem., Int. Ed. Engl. 1996,
35, 1438-1474.
J. Org. Chem, Vol. 71, No. 16, 2006 5857

Khdour et al.
FIGURE 1. Enriched 13C NMR of the methanolic solvolysis products of reduced 1.
SCHEME 7
SCHEME 8
SCHEME 9
of the pyrrolo ring of 5, accounts for the lack of regioselectivity
in nucleophilic attack.
The nucleophile trapping products 21 and 22 are stable
because these systems cannot aromatize. In both products the
leaving group (formally the nucleophile) is either exocyclic or
part of a seven-membered ring. In contrast, nucleophile trapping
by the cyclopropyl quinone methide 5 affords a product that
readily aromatizes to the fused pyrido system, 5 f 6 f 7 in
Scheme 2. Therefore adducts of 2 with biological nucleophiles
were found to be stable (see next section).
In addition to nucleophile trapping, the cyclopropyl quinone
methide 2 traps a proton to afford 19 that is then converted to
18 and 20 by a series of tautomerizations and oxidations,
Scheme 8. The process that converts 19 to reduced 18 (18H2)
is essentially an internal redox reaction wherein electrons flow
from the fused tetrahydropyrido ring to the quinone ring by two
tautomerizations. Aerobic oxidation of 18H2 followed by two
tautomerizations and aerobic oxidation again affords the aro-
matized fused pyrido ring of 20, Scheme 9.
Hydrolysis studies of reduced 1 in pH 7.4 tris buffer afforded
a mixture that was initially analyzed by 13C NMR, Figure 3.
The spectrum reveals the presence of trace starting material 1
and three products identified as 15, 23, and 24, Scheme 10.
The formation of 15 and 23 provide evidence of nucleophile
attack paths illustrated in Figure 2. The dimer 24 must have
resulted from the reaction between two molecules of 2 with
FIGURE 2. Minimized molecular models of the pyrido[1,2-a]indole
cyclopropyl quinone methide reveal the absence of a stereoelectronic
effect on cyclopropane ring opening.
5858 J. Org. Chem., Vol. 71, No. 16, 2006

Cyclopropyl Quinone Methide Alkylating Agent
ing 13C NMR and product isolations. Both nucleophilic and
electrophilic attack on the fused cyclopropane ring of 2 results
in pyrido[1,2-a]indole (ring retention) and azepino[1,2-a]indole
(ring expansion) products. The stereoelectronic effect plays no
discernible role in nucleophilic attack on the flexible pyrido-
[1,2-a]indole system of cyclopropyl quinone methide 2. In
contrast, the stereoelectronic effect dictates nucleophilic attack
in the more rigid pyrrolo[1,2-a]indole system of cyclopropyl
quinone methide 5.10 However, steric effects resulting in
nucleophilic attack occurs at the least hindered cyclopropane
carbon of 2 in the case of a large nucleophile. Nucleophile
trapping products of 2 are stable and do not undergo aromati-
zation, as was the case with nucleophile trapping products of
5. The latter products readily eliminate the nucleophile, and then
air oxidizes to afford a stable aromatic ring.10 Thus the
cyclopropyl quinone methide 2 appears to be a stable alkylation
product suitable for biological systems. Another conclusion is
that the 9-position of the pyrido[1,2-a]indoledione ring system
is susceptible to tautomerization even under mild conditions.
The tautomerization of the C(9) proton to afford a quinone
methide is favored by the six-membered pyrido ring. Water
addition to the quinone methide followed by air oxidation of
the resulting hydroquinone affords the 9-hydroxy derivative.
Similar tautomerization/oxidation reactions have been noted in
other pyrido[1,2-a]indoledione derivatives24 The pyrrolo[1,2-
a]indoledione does not readily undergo tautomerization/oxida-
tion reactions because of the strain of the methide in the five-
membered pyrrolo ring. Currently, we are using the C(9)
oxidation reaction to design novel dual alkylating agents based
on the pyrido[1,2-a]indoledione ring system.
FIGURE 3. Enriched 13C NMR of the hydrolysis products of reduced
1 in pH 7.4 tris buffer.
SCHEME 10
Experimental Section
All analytically pure compounds were dried under high vacuum
in a drying pistol heated with refluxing methanol. Fremy salt was
purchased from a commercial supplier, stored over calcium chloride
desiccant in a refrigerator, and used within a month. Some
compounds contained fractional amounts of water of crystallization
that were determined from the elemental analyses found. Some trace
byproducts were identified on the basis of spectral data without
elemental analyses. Uncorrected melting points and decomposition
points were determined with a Mel-Temp apparatus. All TLC was
run with silica gel plates with fluorescent indicator, employing a
variety of solvents. IR spectra were taken as KBr pellets or thin
films; the strongest IR absorbances are reported. 1H and 13C NMR
spectra were obtained on a 300 MHz spectrometer, and chemical
shifts are reported relative to TMS. The NMR peak assignments
of some compounds were assigned on the basis of two-dimensional
experiments and 13C labeling, and these compounds have peak
assignments.
3,6-Dimethyl-5-methoxyindole (9). Synthesis was carried out
in two steps from the reported compound 825 as follows: To a
solution of 5.0 g (24.2 mmol) of 8 and 20 g (144.7 mmol) of
K2CO3 in 100 mL of dry acetonitrile was added 100 mg of 18-
crown-6 and 2.0 mL of CH3I. The mixture was heated to reflux
and stirred under nitrogen for 15 h. After completion of the reac-
tion (monitored by TLC), the mixture was cooled to room tem-
perature, the salt was filtered off, and the solvent evaporated to a
residue under vacuum. This residue was dissolved into 150 mL of
dichloromethane and filtered again to remove salts. The solvent
one reactant acting as a nucleophile and the other acting as
electrophile. A series of reactions after the coupling, including
tautomerizations, water trapping, water elimination, and oxida-
tions eventually affords the final product 24 as illustrated in
Scheme 11. The presence of a highly extended quinone methide
that traps water by a 1,8-addition reaction (inset of Scheme 11)
accounts for the allylic alcohol of 24.
Cyclopropyl Quinone Methide Trapping of 5′-dGMP. To
assess the trapping of biological nucleophiles, 2 was generated
in the presence of 5′-dGMP. Reductive alkylation of 5′-dGMP
at pH 7.4 afforded a mixture of phosphate adducts 25 and 26
that could not be separated by reverse phase chromatography,
Scheme 12. The 13C NMR spectrum of the purified mixture
shown in Figure 4 reveals that the pyrido[1,2-a]indole 25 was
the major product with trace amounts of azepino[1,2-a]indole
26 present. Since the stereoelectronic effect favors either
product, steric effects must dictate nucleophilic attack at the
least hindered cyclopropane carbon of 2 to afford 25. In contrast,
the nucleophile trapping reactions of cyclopropyl quinone
methide 5 with 5′-dGMP and DNA are controlled exclusively
by the stereoelectronic effect.10
(24) 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.
(25) Macor, J. E.; Wehner, J. M. Tetrahedron Lett. 1991, 32, 7195-
7198.
Conclusions
Cyclopropyl quinone methide 2 formation from the pyrido-
[1,2-a]indoledione ring system 1 has been documented employ-
J. Org. Chem, Vol. 71, No. 16, 2006 5859

Khdour et al.
SCHEME 11
SCHEME 12
of saturated NaCl solution, dried over Na2SO4, and finally filtered
and evaporated to give a thick oily residue that solidified at room
temperature. Recrystallization from ethanol gave light orange
crystals: 4.9 g (92%) yield; mp 79-80 °C; TLC (chloroform) Rf
) 0.47. IR (KBr pellet): 3086, 2966, 2849, 2243 (CN), 1574, 1500
1
cm-1. H NMR (CDCl3): δ 7.97 (1Η, s), 7.10 (1H, s), 4.95 (1H,
q), 3.99 (3H, s), 2.26 (3H, s), 1.71 (3H, d). Anal. Calcd for
C11H12N2O3: C, 59.99; H, 5.49; N, 12.72. Found: C, 60.06; H,
5.52; N, 12.80.
A solution of 5.0 g (22.7 mmol) of methylated 8 in 200 mL of
ethanol was degassed with nitrogen for 5 min. followed by the
addition of 7.15 g of ammonium formate and 1 mL of acetic acid.
To the resulting solution was added 1.5 g of 5% Pd on carbon, and
the reaction was stirred for 45 min at 80 °C and then filtered through
Celite. The solvent was evaporated in vacuo, and the solid residue
was extracted with dichloromethane. The organic layer was washed
with saturated NaCl solution, dried over Na2SO4, filtered, and the
solvent was evaporated to afford the brown crystalline product: 3.1
g (78%) yield; mp 103-104 °C; TLC (chloroform) Rf ) 0.49. IR
(KBr pellet): 3383, 2927, 1472, 1207, 1062 cm-1 1H NMR
.
(CDCl3): δ 7.64 (1H, s), 7.10 and 6.94 (2H, s), 6.87 (1H,d), 3.90
(3H,s), 2.33 (3H, s), 2.30 (3H, s). Anal. Calcd for C11H13NO: C,
75.40; H, 7.48; N, 7.99. Found: C, 75.17; H, 7.44; N, 7.99.
2-Methoxy-3,10-dimethyl-7,8,9a,10-tetrahydro-6H-pyrido[1,2-
a]indol-9-one (10). Synthesis was carried out in two steps from
compound 9 as follows: A stirred mixture of 2.0 g (11.41 mmol)
of 9 in 30 mL of dimethylacetamide, 4.73 g of anhydrous K2CO3,
and 2.0 mL of γ-butyrolactone was heated at reflux under N2 for
48 h. The reaction mixture was diluted with H2O, and the resulting
solution was washed with toluene. The aqueous layer was acidified
with concentrated HCl and then extracted with toluene. The extracts
were evaporated, and the residue was purified by flash chroma-
tography on silica gel using chloroform/ ethyl acetate (1:1) as the
eluent. The isolated product was recrystallized from dichlo-
romethane/hexane: 1.85 g (65%) yield; mp 117-118 °C; TLC
(chloroform, 9:1) Rf ) 0.62. IR (KBr pellet): 2931 (br), 1706, 1478,
was evaporated in vacuo and the residue dissolved in 200 mL of
ethyl acetate and extracted three times with 100 mL portions of
saturated K2CO3. The organic layer was then washed with 100 mL
1
1242 cm-1. H NMR (CDCl3): δ 7.04 (1H, s), 6.91 (1H, s), 6.74
(1H, s), 4.08 (2H,t), 3.88 (3H,s), 2.34 (3H,s), 2.30 (2H,t), 2.27
(3H,s), 2.11 (2H,q).
A mixture of 1.5 g (6.06 mmol) of the above product and 6 g of
polyphosphoric acid was heated at 90 °C for 1 h and then poured
over ice. The resultant slurry was stirred for 4 h, diluted with H2O,
and extracted with Et2O. The combined extracts were washed with
FIGURE 4. Enriched 13C NMR of the GMP trapping products of
reduced 1.
5860 J. Org. Chem., Vol. 71, No. 16, 2006

Cyclopropyl Quinone Methide Alkylating Agent
saturated aqueous NaHCO3, dried with Na2SO4, filtered, and
vacuum-dried. The residue was purified by flash chromatography
on silica gel using dichloromethane as the eluent, and the isolated
product recrystallized from dichloromethane/hexane: 1.35 g (91%)
yield; mp 150-152 °C; TLC (chloroform/ethyl acetate, 90:10) Rf
) 0.44. IR (KBr pellet): 2963, 2918, 1657, 1628, 1535, 1481, 1416,
1339, 1246, 1209 cm-1. H NMR (CDCl3): δ 7.07 and 6.94 (2H,
2s), 4.12 (2H, t, J ) 6 Hz), 3.89 (3H, s), 2.68 (2H, t, J ) 6 Hz),
2.65 (3H, s), 2.36 (3H, s), 2.32 (2H, p, J ) 6 Hz). MS (EI mode)
m/z: 243 (M+) 228 (M+ - CH3), 214 (M+ - CH2CH3), 200. Anal.
Calcd for C15H17NO2: C, 74.05; H, 7.04; N, 5.76. Found: C, 74.10;
H, 7.04; N, 5.7.
6,7,8,9-Tetrahydro-3,10-dimethyl-2-methoxy-1-nitro-9-oxo-
pyrido[1,2-a]indole (11). A mixture of 250 mg (1.03 mmol) of 10
and 25 mL of dichloromethane was cooled at 0 °C. To this mixture
was added 0.20 mL of 69% nitric acid. After this mixture was stirred
for 20 min, the reaction was diluted with 10 mL of saturated
NaHCO3, and the CH2Cl2 layer was separated and dried (Na2SO4).
After removal of the solvent, the crude solid was purified by flash
chromatography on silica gel using chloroform as the eluent. The
isolated product was recrystallized from chloroform/hexane to afford
the product as a yellow crystalline solid: 150 mg (51%) yield; mp
192-194 °C; TLC (chloroform/ethyl acetate, 90:10) Rf ) 0.41. IR
(KBr pellet): 2957, 1751, 1667, 1531, 1329, 1224 cm-1. 1H NMR
(CDCl3): δ 7.25 (1H, s), 4.18 (2H, t, J ) 6 Hz), 3.87 (3H, s), 2.72
(2H, t, J ) 6 Hz), 2.51 and 2.46 (6H, 2s), 2.35 (2H, d, J ) 6 Hz).
MS (EI mode) m/z: 288 (M+), 271 (M+ - OH), 255, 241, 227,
212. Anal. Calcd for C15H16N2O4: C, 62.49; H, 5.59; N, 9.72.
Found: C, 62.55; H, 5.53; N, 9.73.
was warmed to 70 °C for 10 min and then allowed to sit at room
temperature for 24 h. The product was extracted three times
with 25 mL portions of chloroform, and the extracts were dried
(Na2SO4) and concentrated to a residue. The product was purified
by flash chromatography on silica gel using chloroform as the eluant
followed by recrystallization from chloroform/hexane: 120 mg
(72%) yield; mp 85-87 °C; TLC (chloroform) Rf ) 0.37. IR (KBr
pellet): 2953, 2361, 1730, 1523, 1240, 1028 cm-1 1H NMR
.
(CDCl3): δ 7.17 (1H, s), 4.20 and 3.78 (2H, 2m), 4.07 (1H, m),
3.86 (3H, s), 3.70 (3H, s), 2.43 and 2.03 (6H, 2s), 2.38 and 1.99
(2H, 2m), 2.25 and 2.04 (2H, 2m). MS (EI mode) m/z: 315 (M+
- 17), 273, 226, 198, 182. Anal. Calcd for C17H20N2O5: C, 61.44;
H, 6.07; N, 8.43. Found C, 61.53; H, 6.01; N, 8.42. 13C NMR
(CDCl3): δ 172.8 ppm.
6,7,8,9-Tetrahydro-9-(hydroxymethyl)-3,10-dimethyl-2-meth-
oxy-1-nitropyrido[1,2-a]indole(14). To 5 mL of dry THF, cooled
to -20 °C, was added 120 mg (2 mmol) of LAH followed by
addition of a solution of 150 mg of 13 in 5 mL of dry THF over
a 5 min period. The reaction was then stirred for 15 min at -10 to
-20 °C and then diluted with 5 mL of ethyl acetate. The mixture
was filtered through Celite, concentrated to a residue, and purified
by flash chromatography on silica gel using chloroform as the
eluant. The isolated product was recrystallized from chloroform/
hexane: 120 mg (91%) yield; mp 139-141 °C; TLC (chloroform/
methanol, 90:10) Rf ) 0.51. IR (KBr pellet): 3385, 2945, 2884,
2361, 1524, 1462, 1342, 1273, 1154, 1028 cm-1 1H NMR
.
(CDCl3): δ 7.15 (1H, s, aromatic proton), 4.18 and 3.72 (2H, 2m),
3.86 (3H, s), 3.36 (1H, m), 2.43 (3H, s), 2.26 and 1.85 (2H, 2m),
2.11 and 2.02 (2H, 2m), 2.10 (3H, s). MS (EI mode) m/z: 304
(M+), 287 (M+ - OH), 273 (M+ - CH2OH), 256, 241, 226, 198.
Anal. Calcd for C16H20N2O4: C, 63.14; H, 6.62; N, 9.20. Found:
C, 62.90; H, 6.53; N, 9.14. 13C NMR (CDCl3): δ 64.0 ppm.
6,7,8,9-Tetrahydro-9-hydroxymethyl-3,10-dimethyl-2-meth-
oxypyrido[1,2-a]indole-1,4-dione (15). This compound was pre-
pared in two steps: A mixture of 100 mg (0.328 mmol) of 14, 160
mg of 5% pd on carbon, 50 mL of methanol, and 5 drops of acetic
acid was reduced for 2 h under 50 psi H2. The catalyst was removed
by filtration through Celite, and the mixture was concentrated to a
residue. No attempt was made to purify the reactive amino alcohol
product.
9-Cyano-6,7,8,9-tetrahydro-3,10-dimethyl-2-methoxy-1-nitro-
pyrido[1,2-a]indole (12). A solution of 61 mg (0.21 mmol) of 11
and 124 mg (0.64 mmol) of tosylmethyl isocyanide in 2 mL of dry
dimethoxyethane was cooled at 0 °C. To this solution was added
a sodium ethoxide solution freshly prepared by adding 23 mg (1.04
mmol) of sodium to a mixture of 0.25 mL of dry ethanol and 0.35
mL of dry dimethoxyethane. The reaction mixture was stirred for
1 h at 0 °C and then for 1 h at room temperature. The reaction
mixture was diluted with water and acidified with hydrochloric acid
and then extracted three times with 25 mL portions of chloroform.
The organic extracts were dried (Na2SO4 ) and concentrated to a
residue, which was purified by flash chromatography on silica gel
using chloroform as the eluent. The isolated product was recrystal-
lized from chloroform/hexane: 30 mg (48%) yield; mp 154-156
°C; TLC (chloroform/ethyl acetate, 90:10) Rf ) 0.49. IR (KBr
This product was dissolved in 10 mL of water containing 200
mg of monobasic potassium phosphate. To this solution was added
a solution consisting of 500 mg of Fremy Salt in 30 mL of water
containing 300 mg of monobasic potassium phosphate. The reaction
mixture was stirred at room temperature for 18 h and then extracted
four times with 50 mL portions of chloroform. The extracts were
dried (Na2SO4), concentrated to a red solid, and subjected to silica
gel thin-layer chromatographic separation using (1:1) chloroform/
ethyl acetate as the eluent. The isolated product 15 was recrystal-
lized from (chloroform/hexane): 10 mg (10%) yield; TLC (chlo-
roform/methanol, 90:10) Rf ) 0.40. IR (KBr pellet): 3451, 2922,
pellet): 2953, 2924, 2236, 1526, 1462, 1041 cm-1 1H NMR
.
(CDCl3): δ 7.19 (1H, s), 4.26 (1H, m), 4.22 and 3.81 (2H, 2m),
3.87 (3H, s), 2.45 (3H, s), 2.38 and 2.08 (2H, 2m), 2.31 and 2.22
(2H, 2m), 2.15 (3H, s). MS (EI mode) m/z: 299 (M+), 282 (M+
OH), 266, 251, 237, 223. Anal. Calcd for C16H17N3O3: C, 64.20;
H, 5.72; N, 14.04. Found: C, 64.13; H, 5.74; N, 13.97.
-
9-13C-9-cyano-6,7,8,9-tetrahydro-3,10-dimethyl-2-methoxy-1-
nitro-1H-pyrido[1,2-a]indole, 12*. A solution of 30 mg (0.1 mmol)
of 12 and 20 mg (0.4 mmol) of Na13CN in 1 mL of dry DMSO
was heated at 75 °C under N2 for 3 h and then cooled to room
temperature. To the reaction was added 10 mL of water, and the
mixture was extracted three times with 25 mL portions of CHCl3.
The organic extracts were combined and dried with Na2SO4,
concentrated to a residue, and then purified by flash chromatography
on silica gel using chloroform as the eluant. The isolated 12* was
recrystallized from chloroform/hexance: 15 mg (50%) yield.
Incorporation of 13C in the cyanide group was 60% by mass
spectrometry. 13C NMR (CDCl3): δ 118.5 ppm.
2853, 1657, 1632 cm-1 13C NMR (CDCl3) 179.95, 178.21, 156.16,
.
135.14, 129.37, 127.84, 122.24, 118.81, 64.01, 60.89, 45.74, 34.33,
21.72, 19.09, 10.10, 8.66. 1HNMR (CDCl3): δ 4.59 and 4.03 (2Η,
2m), 3.96 (3H, s), 3.74 (2H, m), 3.18 (1H, m), 2.29 (3H, s), 2.18-
1.82 (4H, m), 1.94 (3H, S). MS (EI mode) m/z: 289 (M+), 258,
243, 215, 195. Anal. Calcd for C16H19NO4‚0.1CHCl3: C, 64.19;
H, 6.39; N, 4.65. Found: C, 64.75; H, 6.58; N, 4.88. 13C NMR
(CDCl3): δ 64.01 ppm.
6,7,8,9-Tetrahydro-9-hydroxymethyl-9-hydroxyl-3,10-dimeth-
yl-2-methoxypyrido[1,2-a]indole-1,4-dione (16) and 6,7,-Dihy-
dro-9-hydroxymethyl-3,10-dimethyl-2-methoxypyrido[1,2-a]-
indole-1,4-dione (17). In addition to 15 the following compounds
were isolated from the reaction by preparative TLC. 6,7,8,9-
Tetrahydro-9-hydroxymethyl-9-hydroxyl-3,10-dimethyl-2-methoxy-
pyrido[1,2-a]indole-1,4-dione (16): 4 mg (20% yield); TLC
(chloroform/methanol, 90:10) Rf ) 0.19. IR (KBr pellet): 3430,
Methyl 6,7,8,9-Tetrahydro-3,10-dimethyl-2-methoxy-1-nitro-
pyrido[1,2-a]indole-9-carboxylate (13). To a mixture of 151 mg
(0.51 mmol) of 12 in 8 mL of methanol cooled to -20 to -40 °C
was added hydrogen chloride gas until saturated. The resulting
mixture was stoppered and stored in the freezer for 12 h and then
in a refrigerator for 10 h. The reaction mixture was concentrated
to a residue and combined with 10 mL of 3N HCl. This mixture
1
2928, 1640 cm-1. H NMR (CDCl3): δ 4.49 and 4.06 (2H, 2m,
6-diastereomeric methylene protons), 3.95 (3H, s, methoxy), 3.90
J. Org. Chem, Vol. 71, No. 16, 2006 5861

Khdour et al.
and 3.68 (2H, 2m, methylene protons), 2.46 and 1.92 (6H, 2s, 3,
10-dimethyl), 2.38 ∼ 1.75 (4H, m, 7,8-diastereomeric methylene
protons). Anal. Calcd for C16H19NO4‚0.1H2O: C, 62.57; H, 6.25;
N, 4.55. Found C, 62.61; H, 6.26; N, 4.56. 13C NMR (CDCl3) δ
65.9 ppm.
6,7,-Dihydro-9-hydroxymethyl-3,10-dimethyl-2-methoxypyrido-
[1,2-a]indole-1,4-dione (17): 1 mg (5%) yield; TLC (chloroform/
methanol, 90:10) Rf ) 0.38. IR (KBr pellet): 3534, 2924, 1655,
1628 cm-1; 1H NMR (CDCl3): δ 6.06 (1H, t, J ) 5.1 Hz, 8-alkene
proton), 4.53 (2H, s, methylene protons), 4.47 (2H, t, J ) 7.2 Hz,
8-methylene protons), 3.99 (3H, s, methoxy), 2.53 and 1.95 (6H,
2s, 3, 10-dimethyl), 2.47 (2H, q, J ) 5.1 Hz, 7-methylene protons).
MS (EI mode) m/z: 287 (M+), 272, 195, 153, 137, 116.
of products are provided below. 3,9,10-Trimethyl-2-methoxy-8,9-
dihydropyrido[1,2-a]indole-1,4-dione (18). A total of 0.3 mg (3%)
yield; TLC (chloroform), Rf ) 0.42. H NMR (CDCl3): δ 5.71
1
(1H, m, alkene proton), 4.43 (2H, t, J ) 7.2 Hz, 6-diastereomeric
methylene protons), 3.99 (3H, s, methoxy), 2.52, 2.16, 1.95 (9H,
3s, 3, 9, 10-trimethyl), 2.39 (2H, m, 7-diastereomeric methylene
protons). MS (El mode) m/z: 271 (M+), 256 (M+ - CH3), 241,
228. 3,9,10-Trimethyl-2-methoxy-6,7,8,9-tetrahydropyrido[1,2-
a]indole-1,4-dione (19). A total of 0.8 mg (7%) yield; TLC
(chloroform), Rf ) 0.51. 1H NMR (CDCl3): δ 4.57 (2H, m,
6-diastereomeric methylene protons), 3.96 (3H, s, methoxy), 3.13
(3H, m, methine proton), 2.27, 1.94 (6H, 2s, 3, 10-dimethyl), 2.04,
1.90, and 1.71 (4H, 3m, 7, 8-diastereomeric methylene protons),
1.25 (3H, t, J ) 7.2 Hz). MS (El mode) m/z: 273 (M+), 258 (M+
- CH3), 230, 215. 3,9,10-Dimethyl-2-methoxypyrido[1,2-a]in-
dole-1,4-dione (20). A total of 0.7 mg (6.5%) yield; TLC
(chloroform), Rf ) 0.40 .1H NMR (CDCl3): δ 9.40 (1H, d, J ) 6
Hz, aromatic proton from pyrido ring), 6.82 (1H, m, aromatic proton
from pyrido ring), 3.98 (3H, s, methoxy), 2.85, 2.68 and 2.05 (9H,
6,7,8,9-Tetrahydro-9-hydroxymethyl-9-hydroxyl-3,10-dimeth-
yl-2-methoxypyrido[1,2-a]indol-1-imin,4-one(imino-16). This com-
pound was prepared in two steps: A mixture of 100 mg (0.328
mmol) of 14, 160 mg of 5% Pd on carbon, 50 mL of methanol
was reduced for 2 h under 50 psi H2.The catalyst was removed by
filtration through Celite and concentrated to a residue. No attempt
was made to isolate the reactive amino alcohol product. This product
was dissolved in 3.0 mL acetone. To this a solution of phosphate
buffer pH 7.2 with 700 mg of Fremy salt was added. The reaction
mixture was stirred at room temperature for 15 min and then
extracted four times with 50 mL of chloroform. The extracts were
dried (Na2SO4), concentrated to a yellow solid, and subjected to
silica gel thin-layer chromatographic separation using (1:1) chlo-
roform/ ethyl acetate as the eluent. The isolated product imino-16
was recrystallized from (chloroform/hexane): 52 (52%) yield; mp
179 > (dec); TLC (ethyl acetate) Rf ) 0.33. IR (KBr pellet): 3414,
3s, 3, 9, 10-trimethyl). MS (El mode) m/z: 269 (M+), 254 (M+
-
CH3), 226. 3,10-Dimethyl-2-methoxy-9-methoxymethyl-6,7,8,9-
tetrahydropyrido[1,2-a]indole-1,4-dione (21). A total of 2 mg
(16%) yield; TLC (chloroform), Rf ) 0.29. IR (KBr pellet): 2924,
1661, 1636, 1203, 1456 cm-1. 1H NMR (CDCl3): δ 4.63 and 3.96
(2H, 2m, 6-diastereomeric methylene protons), 3.97 (3H, s, meth-
oxy), 3.42 (2H, m, methylene protons), 3.37 (3H, s, methoxy), 3.26
(1H, s, methine proton), 2.29 and 1.94 (6H, 2s, 3, 10-dimethyl),
2.13, 2.02 and 1.76 (4H, 3m, 7, 8-diastereomeric methylene
protons). MS (El mode) m/z: 303 (M+), 258 (M+ - CH2OCH3),
243, 226. 3,10-Dimethyl-2,9-dimethoxy-6,7,8,9-tetrahydroaze-
pino[1,2-a]indole-1,4-dione (22). A total of 2.5 mg (20%) yield;
TLC (chloroform), Rf ) 0.28. 1H NMR (CDCl3): δ 4.86 and 4.49
(2H, 2m, 6-diastereomeric methylene protons), 3.98 (3H, s, meth-
oxy), 2.91 (2H, m, 10-diastereomeric methylene protons), 3.36 (3H,
s, methoxy), 2.29 and 1.94 (6H, 2s, 3, 10-dimethyl), 2.05, 1,88
and 1.67 (4H, 3m, 7, 8-diastereomeric methylene protons). MS
3242, 2936, 2860, 1639, 1616, 1587 cm-1 13C NMR (DMSO):
.
177.13, 159.08, 153.49, 138.51, 124.88, 124.79, 122.92, 117.33,
1
70.65, 65.71, 61.31, 45.35, 31.64, 19.58, 11.76, 8.97. H NMR
(DMSO): δ 10.76 (1H, s), 5.16 (1H, s), 4.85 (1H, t), 4.29 and
4.14 (2H, 2m), 3.72 (3H, s), 3.62 and 3.53 (2H, 2m), 2.49 (3H, s),
2.21 (1H, m), 2.34 (2H, 2m), 1.88 (3H, s), 1.62 (1H, m). MS
(APCI+ mode) m/z: 305.1503. MALDI m/z: calculated, 305.150
(M + 1); found, 305.152 (M + 1). Anal. Calcd for C16H20N2O4‚
0.1CHCl3: C, 61.14; H, 6.41; N, 8.86. Found: C, 60.78; H, 6.34;
N, 8.64.
(El mode) m/z: 303 (M+), 288 (M+ - CH3), 272, 258 (M+
CH2OCH3), 245.
-
Hydrolysis of 1H2 in Anaerobic Aqueous Buffer. A solution
consisting of 0.5 mL DMSO and 5 mg (0.06 mmol) of 1 (13C
enriched) was added to 2 mL of 0.05 M pH 7.4 tris buffer containing
1 M KCl. To this solution was added 3 mg of 5% Pd on carbon,
and the mixture was then degassed with argon for 30 min, followed
by bubbling hydrogen gas for 15 min, and finally bubbling with
argon for 30 min to remove the excess hydrogen. The reaction
mixture was incubated at 30 °C for 24 h and then opened to the
air. The catalyst was filtered off, and the filtrate extracted three
times with 20 mL portions of chloroform. The extracts were dried
(Na2SO4) and concentrated to a red solid, which was subjected to
preparative silica gel thin-layer chromatographic separation using
chloroform/methanol (95:5) as eluant. The physical properties of
hydrolysis products are provided below.
6,7,8,9-Tetrahydro-3,10-dimethyl-9-[(methanesulfonoxy)-
methyl]-2-methoxypyrido[1,2-a]indole-1,4-dione (1). A total of
0.1 mg (2%) yield. 13C NMR (CDCl3): δ 68.4 ppm.
6,7,8,9-Tetrahydro-9-hydroxymethyl-3,10-dimethyl-2-meth-
oxypyrido[1,2-a]indole-1,4-dione (15). A total of 1.2 mg (30%)
yield. 13C NMR(CDCl3): δ 63.7 ppm.
6,7,8,9-Tetrahydro-3,10-dimethyl-9-[(methanesulfonoxy)-
methyl]-2-methoxypyrido[1,2-a]indole-1,4-dione (1). To a stirred
of solution of 15 mg of 15 in 0.3 mL of dry pyridine, cooled to 0
°C, was added 0.06 mL of methanesulfonyl chloride. After the
reaction was stirred for 20 min at 0 °C and then 1.5 h at room
temperature, the reaction mixture was diluted with a mixture
consisting of 10 mL chloroform, and 10 mL of water was then
added dropwise to decompose the excess methanesulfonyl chloride.
The chloroform layer was washed consecutively with 2 N HCl,
5% sodium bicarbonate, and finally water. The chloroform layer
was dried over Na2SO4 and then concentrated to an orange solid,
which was recrystallized from chloroform/ hexane): 16 mg (84%)
yield; mp 103-104 °C; TLC (chloroform/methanol, 90:10); Rf )
0.69. IR (KBr pellet): 3634, 2942, 1655, 1636, 1611, 1346, 1171
1
cm-1. H NMR (CDCl3): δ 4.67 and 3.96 (2H, 2m), 4.31 and
4.18 (2H, 2m), 3.98 (3H, s), 3.46 (1H, m), 3.02 (3H, s), 2.30 (3H,
s), 2.17 and 1.79 (2H, 2m), 2.00 (2H, m), 1.94 (3H, s). MS (EI
mode) m/z: 367 (M+), 271 (M+ - MsOH), 258 (M+ - CH2OMs),
243, 228, 215. Anal. Calcd for C17H21NO6S: C, 55.57; H, 5.76;
N, 3.81. Found C, 55.55; H, 5.76; H, 3.81. 13C NMR (CDCl3): δ
68.4 ppm.
3,10-Dimethyl-9-hydroxy-2-methoxy-6,7,8,9-tetrahydroazepino-
[1,2-a]indole-1,4-dione (23). A total of 0.7 mg (18%) yield. 13C
NMR (CDCl3): δ 32.4 ppm.
Hydrolysis of Reduced 1 in Anaerobic Methanol. To a mixture
consisting of 15 mg (0.04 mmol) of 1 and 6 mg of 5% Pd on carbon
was added 8 mL of methanol. The mixture was bubbled with argon
for 15 min, with hydrogen gas for 10 min, and finally with argon
for 15 min. The reaction mixture was incubated at 30 °C for 24 h
and then combined with air and stirred for 1 h. The catalyst was
filtered off, and the filtrate was concentrated to a red solid, which
was subjected to preparative silica gel thin-layer chromatographic
separation using chloroform as the eluant. The physical properties
Dimer (24). A total of 0.4 mg (5%) yield. MS (El mode) m/z:
572, 556, 512, 496. 13C NMR (CDCl3): δ 44.8 ppm.
Preparation of the 5′-dGMP Adduct (25). To a solution of 35
mg (0.076 mmol) of the disodium salt of 5′-dGMP‚3H2O in 5 mL
of 0.05 M pH 7.4 tris‚HCl, containing 5 mg of 5% Pd on carbon,
was added a solution of 7 mg (0.14 mmol) of 1 in 0.5 mL of DMSO.
The mixture was deaerated with argon for 30 min and then purged
with H2 gas until the reaction mixture became colorless, about 10
5862 J. Org. Chem., Vol. 71, No. 16, 2006

Cyclopropyl Quinone Methide Alkylating Agent
min. The excess H2 was then removed by purging with argon for
10 min. The reduced reaction mixture was incubated at 30 °C for
24 h and then opened to the air. The reaction mixture was
centrifuged at 12 000 g for 20 min, and the supernatant was
extracted three times with chloroform to remove hydrolysis
products. TLC analysis of the hydrolysis products revealed the
presence of 6,7,8,9-tetrahydro-9-hydroxymethyl-3,10-dimethyl-2-
methoxypyrido[1,2-a]indole-1,4-dione (15) (13C NMR (CDCl3): δ
63.7 ppm) and 3,10-dimethyl-9-hydroxy-2-methoxy-6,7,8,9-tet-
rahydroazepino[1,2-a]indole-1,4-dione (23) (13C NMR (CDCl3): δ
32.4 ppm).
The 5′-dGMP moved somewhat faster than 25 and 26 while eluting
with water. The orange band was collected and then concentrated
to a dry residue and dissolved in 1 mL of water. The small amount
of column residue was removed by centrifugation (12 000 g 10
1
min), and the supernant lyophilized to a red solid. HMR (D2O):
δ 8.14 (1H, bs, amide proton from guanine), 7.90 (1H, s, C(8)
proton from guanine), 6.56 (2H, bs, amine protons from guanine),
6.10 (1H, t, J ) 7 Hz, C-1′ proton from deoxy-ribose), 4.43 (2H,
m), 4.00 and 3.70 (2H, 2m), 3.83 (3H, s, methoxy), 3.80 (1H, s),
2.16 and 1.81 (6H, 2s). 13C NMR (D2O): δ 64.10 ppm
The aqueous phase was orange in color and contained 25 and
26 along with unreacted 5′-dGMP. Concentration of the aqueous
phase to complete dryness (no DMSO remaining) under high
vacuum was followed by dissolution in ∼1 mL of H2O and then
placement on a 100 g reverse phase column prepared with water.
Acknowledgment. We thank the National Science Founda-
tion for their generous support.
JO0602423
J. Org. Chem, Vol. 71, No. 16, 2006 5863