Practical synthesis of a potent indolocarbazole-based, DNA topoisomerase inhibitor

Article abstract of DOI:10.1016/S0040-4020(01)00895-X

DNA topoisomerase I inhibitors are currently under investigation as cancer chemotherapy agents of which indolocarbazole glycoside (1) has been identified as a promising candidate. A practical, scalable synthesis of 1 that limits the isolation of cytotoxic compounds to only that of the final product is described. The convergent process features a novel phase transfer-promoted glycosylation of aglycone core (4); subsequent hydrolysis provides anhydride (8). The hydrazine fragment (3), which is coupled with 8, is synthesized via a modification of existing procedures. The coupled product (2) is subsequently hydrogenated to provide 1 in excellent purity via direct crystallization (>99.3 A%).

Full text of DOI:10.1016/S0040-4020(01)00895-X

TETRAHEDRON
Pergamon
Tetrahedron 57 .2001) 8917±8923
Practical synthesis of a potent indolocarbazole-based,
DNA topoisomerase inhibitor
Atsushi Akao,a,p Shouichi Hiraga,a Takehiko Iida,a Asayuki Kamatani,a Masashi Kawasaki,a
Toshiaki Mase,a Takayuki Nemoto,a Nobuya Satake,a Steven A. Weissman,b,p David M. Tschaen,b
Kai Rossen,b Daniel Petrillo,b Robert A. Reamerb and R. P. Volanteb
aProcess R & D, Banyu Pharmaceutical Co. Ltd, Okazaki, Aichi 444-0858, Japan
bProcess Research, Merck and Co. Inc., Rahway, NJ 07065, USA
Received 8 August 2001; accepted 6 September 2001
AbstractÐDNA topoisomerase I inhibitors are currently under investigation as cancer chemotherapy agents of which indolocarbazole
glycoside .1) has been identi®ed as a promising candidate. A practical, scalable synthesis of 1 that limits the isolation of cytotoxic
compounds to only that of the ®nal product is described. The convergent process features a novel phase transfer-promoted glycosylation
of aglycone core .4); subsequent hydrolysis provides anhydride .8). The hydrazine fragment .3), which is coupled with 8, is synthesized via a
modi®cation of existing procedures. The coupled product .2) is subsequently hydrogenated to provide 1 in excellent purity via direct
crystallization ..99.3 A%). q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
practical route to 1 in support of developmental studies as
the existing route5 was not amenable to larger scale syn-
thesis. The overriding strategy in this second-generation
synthesis was to minimize the handling of topoisomerase-
active intermediates. As the chemistry in the early steps
dictated using fully protected compounds, coupled with
the knowledge that the perbenzylated analog of 1 was not
topo I active,6 we sought to remove the protecting groups as
the last step of the synthesis. Thus, the perbenzylated analog
.2) became our penultimate target.
The development of DNA topoisomerase I .topo I) inhibi-
tors as cancer chemotherapy agents is currently an active
area of research.1 Speci®cally, the indolocarbazole glyco-
side class of compounds, such as the rebeccamycin
analogs,2 have been identi®ed as attractive clinical targets.3
Among them, 1 has emerged based on its potent cytotoxic
activity against human cancer cells4 and its wide safety
margin and is currently in clinical trials. We required a
Scheme 1.
Keywords: indolocarbazole; topoisomerase; glycosylation.
Corresponding authors. Tel.: 11-732-594-3589; fax: 11-732-594-1499; e-mail: steven_weissman@merck.com
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020.01)00895-X

8918
A. Akao et al. / Tetrahedron 57 /2001) 8917±8923
Scheme 2. Reagents and conditions: .a) thionyl chloride, DMF 5±258C/2 h ..95%); .b) 4, 48% aqueous KOH, Aliquat 336, MTBE, rt/3 h .83%); .c) 48%
KOH, EtOH, toluene, 308C/16 h; .d) Aqueous citric acid to pH 8, 258C/6 h .82%); .e) 3a, TEA, DMA, 658C/3 h .99%); .f) H2 .40 psi), 10% Pd/C, THF, IPA,
aqueous HCl, 408C/14 h .82%).
A retrosynthetic analysis of 2 reveals 1,3-dibenzyloxy-2-
hydrazinopropane .3), indolocarbazole core .4)5 and
perbenzylated glucopyranose derivative 5 as accessible
building blocks. Herein, we disclose a practical, convergent
synthesis of 1 from readily-available starting materials
utilizing this strategy .Scheme 1).
related systems is known,7 to our knowledge there are no
examples using indolocarbazole substrates in an aqueous
medium. Accordingly, after treatment of 5 with thionyl
chloride in DMF and subsequent isolation of the glycosyl
chloride in MTBE, a series of common phase transfer
catalysts conditions were screened in the reaction with
indolocarbazole 4. Subsequently, the use of Aliquat 336
.tricaprylylmethylammonium chloride; 1.4 equiv.) in
combination with 48% aqueous KOH and MTBE .258C,
2.5 h) provided an 83% yield of glycoside 6 after crystal-
lization from MTBE/MeOH. The ratio of anomers was
.150:1 .b:a). The use of sub-stoichiometric amounts of
Aliquat 336 led to incomplete reactions. Although the role
of this reagent is not clear, we postulate that the ®rst
equivalent forms a complex with 4, whereas the remainder
works as a phase transfer catalyst; for upon addition of
1.0 equiv. Aliquat 336 to a slurry of 4 in MTBE in the
presence of base, an orange powder precipitates whose
solid-state NMRdiffers from that of 4. Indeed, addition of
base and glycosyl chloride to this mixture does not initiate
the reaction; further addition of Aliquat 336 is necessary.
The formation of the di-glycoside was not observed under
these Aliquat 336-promoted conditions .Scheme 2).
2. Results and discussion
2.1. Glycosylation/hydrolysis
The direct glycosylation of indolocarbazoles remains a
formidable challenge due to the low nucleophilicity of the
nitrogen atoms and the necessity for high levels of stereo-
selectivity. Prior glycosylations of protected indolocarba-
È
zoles have utilized Mitsunobu or Konigs±Knorr .silver or
mercury-based reagents) conditions2b,4,5 to achieve the
necessary levels of stereoselectivity. These approaches
suffer from low-modest yields, and the inclusion of
undesirable reagents and/or by-products. The use of
powdered KOH, tert-BuOK and other strong bases under
anhydrous conditions has been demonstrated to provide
the b-N-glycosides in high yield as well.4 The need to
prepare the former reagent immediately prior to use and
the extreme water sensitivity of the reactions rendered
them less attractive. Alternatively, we sought to implement
phase transfer agents in combination with an aqueous base
to overcome the shortcomings of the prior methodologies.
While the phase transfer-mediated alkylation of indoles and
The conversion of 6 to anhydride 8 was accomplished by
treatment with aqueous KOH in toluene/EtOH for 16 h at
308C. The reaction initially generates a mixture of regio-
isomeric amic acid intermediates .7), which upon adjust-
ment to pH 7.5±8 at 58C with aqueous citric acid
undergoes dehydration to form the anhydride over the

A. Akao et al. / Tetrahedron 57 /2001) 8917±8923
8919
amine at 658C. The triethylamine was added at 658C to
minimize decomposition of 3 to H2NNH2, which can also
couple with 8 to form a N-amino-maleimide impurity. The
cooled reaction mixture was partitioned between water and
MTBE. The inability to crystallize 2 led to its introduction
into the next step as a THF solution .quantitative yield) after
a solvent switch from MTBE.
Hydrogenation of 2 in i-PrOH/THF in the presence of
aqueous HCl proceeded to completion ..99.8% conver-
sion) in 4±14 h at 408C with 10% Pd±C to provide 1.
Under these conditions, no N±N bond cleavage was
observed. Removal of the catalyst through solka ¯oc was
suf®cient to minimize residual Pd in the ®nal product
.,15 ppm). The ®ltrate was pH adjusted to 2.5 with 1 M
triethylamine in i-PrOH. Water was then added to the ®ltrate
to avoid precipitation of the amorphous form of 1 during the
subsequent concentration to remove the THF and toluene.
The water content was ®rst lowered to 20% via azeotropic
drying and seed was added. Interestingly, the requisite level
of crystal growth, as measured by supernatant concen-
tration, was achieved only after prolonged aging .9 h) at
708C. The water content was then lowered to 3% by
means of i-PrOH addition and azeotropic drying. Crystalline
1 was isolated in 82% yield on cooling to 228C.
Scheme 3. Reagents and conditions: .a) aqueous NaOCl, cat. TEMPO,
MeCN, 58C/1 h; .b) Boc-NH-NH2, heptane/toluene, 708C/1 h .86% from
9); .c) .i) NaBH4, BF3±etherate, THF, 08C/1 h; .ii) 6N HCl, 608C/4 h;
.iii) 0.5 equiv. oxalic acid, MTBE, EtOH, 20±408C/12 h .79% overall).
course of 6 h at 258C. This pH adjustment is critical as
failure to keep the pH above 7 led to higher levels of 6
..2 area% by HPLC), the result of the retro reaction.
Extraction into MTBE followed by crystallization from
MeCN/MeOH provided 8 in 82% yield.
2.2. Synthesis of hydrazine oxalate )3a)
2.4. NMR analysis of 1 and related compounds
The synthesis of the hemioxalate salt of 3 .3a), based on a
prior methodology,8 began with conversion of commer-
cially-available 1,3-dibenzyloxy-2-propanol .9) to ketone
10 via a TEMPO-catalyzed oxidation with bleach as the
co-oxidant in MeCN/aqueous NaHCO3.9 The use of the
NaHCO3 buffer was critical to avoid over-oxidation of rela-
tively unstable 10. The ketone was carried forward into the
next step as a slurry in heptane, obviating the need for iso-
lation, whereupon it was treated with a solution of Boc-
hydrazine in toluene. Condensation was complete within
3 h at 708C to afford hydrazone 11 in 85% overall yield
from 9 upon direct crystallization from the cooled reaction
mixture .Scheme 3).
Interest in the conformational control of the indolocarbazole
glycosides, which stems from the theory that it plays a role
in pre-organizing the molecule for biological activity,10 led
us to study the solution-phase behavior of 1. 1H NMRanaly-
sis of 1, in DMSO-d6, shows the presence of two conformers
.95/5 ratio) corresponding to rotational isomers arising from
restricted bond rotation between the indolo nitrogen and the
anomeric carbon of the sugar. Gradient-enhanced NOE
.Nuclear Overhauser Effect) experiments11 were used to
assign these rotamers and the diagnostic NOEs are shown
in Fig. 1.
Similar NMRstudies for 2 show the same major rotamer
although the ratio has become 82/18. Essentially the same
rotamer distribution is also seen for 6 in DMSO-d6.
However, when 6 is run in CDCl3 there is only one rotamer
observed. NOE studies show that this rotamer is the same as
the major rotamer seen in DMSO-d6 solution. The solvent
dependance of the rotameric ratio is due to the ability of
DMSO to hydrogen bond with the NH proton, whereas
chloroform cannot. The strong preference for the observed
conformation .`closed') in glycosides 1, 2 and 6 is attributed
to hydrogen bonding between the indole NH and the
ethereal oxygen of the pyranose, a common feature in this
class of molecules.10
Subsequent reduction of hydrazone 11 with commercial lots
of borane±THF suffered from variable yields, but in-situ
formation from sodium borohydride and boron tri-
¯uoride±etherate .1.5 equiv.) in THF proved more reliable.
The reaction proceeded to completion in 1 h at 0±58C.
Subsequent treatment with 6N aqueous HCl, followed by
heating to 658C for 3 h served to quench the reaction and
remove the Boc group. Following neutralization, the
product was crystallized as the hemioxalate salt 3a from a
MTBE/THF/EtOH solvent system at 408C, giving colorless
plates in 79% overall yield. It is noteworthy that the free
base of 3a decomposes upon exposure to air, forming
H2NNH2 that complicates the subsequent steps. Thus the
use of deoxygenated/degassed solvents and handling under
a nitrogen atmosphere were necessary upon extraction until
the salt formation.
3. Conclusion
We have developed a convergent, readily-scalable synthesis
of 1 in 56% overall yield from 4 which minimizes the iso-
lation of cytotoxic intermediates to only that of the ®nal
compound. Highlights of the process include the use of a
highly stereoselective, phase transfer-promoted glycosyl-
ation and the direct crystallization of 1 from the reaction
2.3. Endgame-synthesis of 1
The coupling of hydrazine 3a with anhydride 8 was run in
deoxygenated dimethylacetamide .DMA) with triethyl-

8920
A. Akao et al. / Tetrahedron 57 /2001) 8917±8923
below 108C. The resulting mixture .pH 12.5) was warmed to
258C, and the organic layer separated and washed with
water .2£38 L) and 25% brine .27 L). The organic layer
was then diluted with MTBE to make a 70-L solution and
1
used as is in the next step. H NMRwas consistent with
previously reported values.12
4.1.2. Synthesis of indolocarbazole glycoside )6). The
MTBE solution .70 L) of 2,3,4,6-tetra-O-benzyl-d-gluco-
pyranose chloride was added to a well-dispersed suspension
of 45 .2,10-dibenzyloxy-12,13-dihydro-5H-indolo[2,3-a]-
pyrrolo[3,4-c]carbazole-6-methyl-5,7-.6H)-dione) .10.0 kg,
18.1 mol), MTBE .70 L) and 48% aqueous KOH .50 L) in a
600-L tank at 258C. After 0.5 h, 48% aqueous KOH .50 L)
was added to the vessel. The resulting biphasic yellow slurry
was stirred for 30 min at 22±258C, followed by the rapid
addition of a solution of Aliquat 336 .10.3 kg, 25.4 mol) in
MTBE .50 L) to the mixture. The resulting reddish-brown
suspension was further stirred at 20±258C for 2.75 h, at
which time the color became reddish-purple with increased
viscosity. The organic layer was separated, washed with
water .50 L) and cooled below 58C. 1N aqueous HCl
.20 L) was then added slowly to the mixture, keeping the
internal temperature below 158C. The resulting biphasic
mixture was warmed to 20±258C, and the pH adjusted to
1.5±2.5 with additional 1N aqueous HCl .1 L). The organic
layer was separated and washed with water .50 L) and 25%
brine .50 L). The ®ltered organic solution was concentrated
in vacuo to the 140-L level, and MeOH .20 L) was added to
the mixture at 20±238C followed by a seed of product,
which initiated crystallization. The mixture was aged for
2 h at 20±258C, followed by the addition of MeOH .80 L)
over 1 h. The resulting suspension was aged at room
temperature overnight, and the crystals were isolated by
®ltration, washed with a 4:1 .v/v) mixture of MeOH/
MTBE .150 L) and dried in vacuo to afford 6 .16.2 kg,
83%, 99.4 area% by HPLC) as a yellow powder: mp 114±
1158C; 1H NMR.400.25 MHz, CDCl 3-selected data) d
10.67 .s, 1H), 9.25 .d, Jˆ9.2 Hz, 1H), 9.14 .d, Jˆ9.2 Hz,
1H), 7.03 .m, 1H), 6.91 .t, Jˆ7.6 Hz, 2H), 6.22 .d, Jˆ
7.6 Hz, 2H), 5.86 .d, Jˆ8.8 Hz, 1H), 5.19 .overlapping m,
3H), 5.09 .d, Jˆ11.6 Hz, 1H), 4.99 .d, Jˆ10.4 Hz, 1H), 4.90
.d, Jˆ10.8 Hz, 1H), 4.87 .d, Jˆ10.8 Hz, 1H), 4.76 .d, Jˆ
13.1 Hz, 1H), 4.68 .d, Jˆ10.4 Hz, 1H), 4.58 .d, Jˆ13.1 Hz,
1H), 4.34 .t, Jˆ9.6 Hz, 1H), 4.04 .overlapping m, 2H), 3.93
.overlapping m, 2H), 3.88 .d, Jˆ9.6 Hz, 1H), 3.81 .dd,
Jˆ10.0, 2.0 Hz, 1H), 3.32 .s, 3H), 3.01 .d, Jˆ9.6 Hz,
1H); 13C NMR.100.61 MHz, CDCl 3) d 170.3, 170.2,
159.3, 159.3, 143.1, 142.9, 137.9, 137.6, 136.9, 136.9,
136.7, 136.1, 130.0, 128.7, 128.6, 128.5, 128.4, 128.3,
128.2, 128.2, 128.1, 128.0, 128.0, 127.9, 127.9, 127.8,
127.7, 127.7, 127.6, 127.3, 126.7, 126.4, 120.1, 119.3,
118.9, 118.2, 116.8, 116.7, 110.3, 96.6, 96.3, 85.7, 84.7,
80.8, 76.0, 75.9, 75.4, 74.8, 73.9, 70.6, 70.4, 66.7, 23.7;
HRMS .FAB) 1073.4242 .M, 1073.4251 calcd for
C69H59N3O9).
Figure 1.
mixture of suf®cient purity ..99.3 area% by HPLC) to
obviate the need for further puri®cation. NMRanalysis
indicates that indolocarbazole glycosides described herein
adopt the expected `closed' conformation.
4. Experimental
4.1. General methods
All reactions were carried out under a nitrogen atmosphere.
All commercial chemicals were used without further
puri®cation; 2,3,4,6-tetra-O-benzyl-d-gluco-pyranose and
Aliquat 336 were purchased from Sigma/Aldrich. 1,3-
Dibenzyloxy-2-propanol was purchased from Dixie
Chemical .Texas, USA). Degassed solvents were prepared
by placing under vacuum followed by purging with nitrogen
or by sparging with nitrogen. Indolocarbazole 4 was
prepared according to Ref. 5. NMR spectra were recorded
on Bruker AM-300, DPX-400 and DRX-600 spectrometers.
Spin±spin coupling constants .J) are reported in Hertz.
4.1.1. Chlorination of 2,3,4,6-tetra-O-benzyl-d-gluco-
pyranose )5). SOCl2 .3.71 kg, 31.2 mol) was added slowly
to a chilled solution of 2,3,4,6-tetra-O-benzyl-d-gluco-
pyranose .5) .14.7 kg, 27.2 mol) and DMF .59 L) in a
80-L glass vessel, keeping the internal temperature below
58C. The resulting mixture was warmed to 25±308C and
aged for 2 h. MTBE .66 L) was added to the vessel followed
by cooling below 08C. 0.5N aqueous NaOH .210 L) was
slowly added to the vessel, keeping the internal temperature
4.1.3. Synthesis of anhydride )8). EtOH .145 L) was added
over 30 min to a biphasic mixture of 6 .16.1 kg, 14.9 mol),
toluene .58 L) and 48% aqueous KOH .43.1 kg) in a 600-L
tank at 208C, keeping the internal temperature below 308C.
The resulting dark red mixture was stirred at 20±308C for
16 h, during which time the mixture became a homogeneous

A. Akao et al. / Tetrahedron 57 /2001) 8917±8923
8921
red solution. The mixture was aged at 2108C for 1 h,
followed by the slow addition of 10% aqueous citric acid
.240 L), keeping the internal temperature below 58C. The
resulting mixture .pH 7.7±8.0) was stirred at 25±308C for
6 h, during which time an additional 10% aqueous citric
acid .22 kg) was periodically added to maintain pH at
7.5±8.0. The mixture was then extracted with MTBE
.135 L), and the separated organic layer was washed with
3% aqueous NaCl .2£33 L) and 25% aqueous NaCl .33 L),
and treated with carbon .Darco G-60, 1.61 kg, room
temperature, 1 h). The ®ltered solution was concentrated
in vacuo to the 70 L level, and MeCN ¯ushes .2£70 L)
were performed, each time concentrating in vacuo to a
70 L batch volume .residual toluene: 3.6%). The mixture
was then diluted with MeCN to make a 240 L solution,
and MeOH .66 L) was added slowly over 30 min at 22±
258C followed by a seed of product, which initiated crystal-
lization. The resulting slurry was aged at this temperature
range for 1 h, followed by the slow addition of MeOH
.257 L) over 1 h. The resulting yellow suspension was
aged at 22±258C for 1 h followed by aging at 0±58C for
2 h. The crystals were isolated by ®ltration, washed with a
9:1 .v/v) mixture of MeCN/MeOH .2£60 L) and dried in
vacuo to afford 8 .13.1 kg, 82%, 99.3 area% by HPLC) as a
yellow powder: mp 95±968C; 1H NMR.400.13 MHz,
DMSO-d6-selected data-major rotamer) d 11.06 .s, 1H),
8.80 .d, Jˆ8.8 Hz, 1H), 8.67 .d, Jˆ8.8 Hz, 1H), 7.74 .d,
Jˆ2.0 Hz, 1H), 6.97 .m, 1H), 6.81 .m, 2H), 6.62 .d, Jˆ
9.2 Hz, 1H), 6.11 .m, 2H), 5.26 .d, Jˆ11.6 Hz, 1H), 5.21
.s, 2H), 5.00 .d, Jˆ11.6 Hz, 1H), 4.91 .d, Jˆ10.8 Hz, 1H),
4.90 .d, Jˆ13.8 Hz, 1H), 4.81 .s, 2H), 4.66 .d, Jˆ13.8 Hz,
H), 4.62 .d, Jˆ11.6 Hz, 1H), 4.30 .m, 2H), 4.12 .m, 1H),
3.90 .overlapping m, 4H); 13C NMR.100.61 MHz, DMSO-
d6-major rotamer) d 165.0, 164.9, 159.7, 159.5, 143.7,
142.7, 138.5, 138.4, 137.6, 137.1, 137.1, 136.7, 130.4,
129.0, 129.0, 128.9, 128.8, 128.7, 128.6, 128.6, 128.6,
128.4, 128.4, 128.3, 128.2, 128.2, 128.0, 127.9, 127.8,
127.8, 127.7, 127.6, 127.1, 125.1, 125.0, 119.0, 118.2,
118.2, 116.7, 115.6, 115.2, 111.8, 111.3, 97.7, 96.7, 84.8,
83.3, 81.1, 76.6, 76.4, 75.3, 74.7, 74.0, 73.3, 70.5, 69.9,
66.9; HRMS .FAB) 1060.3937 .M, 1060.3935 calcd for
C68H56N2O10).
1 h, followed by cooling to 608C. A seed of product was
added, and the resulting mixture was aged at 608C for 1 h to
initiate crystallization. The mixture was cooled to room
temperature and aged overnight. The crystals were isolated
by ®ltration, washed with n-heptane .10.3 kg) followed by a
9:1 v/v mixture of n-heptane/t-BuOH .12.5 L) and n-hep-
tane .2£10.3 kg), and dried in vacuo at 508C to provide 11
.6.05 kg, 86%, 99.98 area% by HPLC) as colorless needles:
mp 92±938C; 1H NMR.400.13 MHz, DMSO- d6) d 9.86 .br
s, 1H) 7.24±7.38 .br m, 10H), 4.49 .s, 2H), 4.46 .s, 2H),
4.31 .s, 2H), 4.06 .s, 2H), 1.43 .s, 9H); 13C NMR
.100.61 MHz, DMSO-d6)) d 152.7, 148.6, 138.4, 137.9,
128.7, 128.1, 128.1, 128.1, 127.9, 80.1, 72.6, 72.0, 65.5,
28.4. Anal. Calcd for C22H28N2O4: C, 68.73; H, 7.34; N,
7.29. Found: C, 68.75; H, 7.33; N, 7.26.
4.1.5. Synthesis of the hemioxalate salt of 1,3-dibenzyl-
oxy-2-hydrazinopropane )3a). A stirred suspension of
NaBH4 .1.326 kg, 35.01 mol) and THF .30 L) was treated
slowly with a solution of hydrazone 11 .6.00 kg, 15.61 mol)
in THF .42 L) in a 200-L tank at 08C, keeping the internal
temperature below 58C. BF3´OEt2 .3.321 kg, 23.40 mol)
was then added dropwise to the resulting mixture, keeping
the internal temperature below 108C. The resulting colorless
suspension was stirred at 0±58C for 1 h .reaction can be
properly monitored by HPLC after mixing the sample
with EtOH and subsequent re¯ux for 4 min to decomplex
boranes from the product), at which time 6N aqueous HCl
.17.09 kg) was added dropwise over 1 h, keeping the inter-
nal temperature below 208C .caution: vigorous gas evolu-
tion). The resulting colorless suspension was warmed to 60±
658C and aged for 3.5 h, at which time the gas evolution
ceased. Degassed 2N aqueous NaOH .50.9 L) was then
added slowly to the mixture at 38C, keeping the internal
temperature below 208C, followed by warming the resulting
mixture to room temperature and extraction with degassed
MTBE .100 L). The separated organic layer was washed
with degassed water .24 L) followed by degassed brine
.26 kg). Toluene .604.9 g) was then added .internal HPLC
standard) to the organic layer, followed by stirring the
resulting mixture for 5 min. The concentration of the free
base measured by a potentiometric titration: 26.6 mg/mL so
that 4.12 kg .14.4 mol) of free base was estimated in the
mixture .batch volumeˆ155 L). The organic layer was
then diluted with degassed MTBE to form a 180-L solution.
Then EtOH .90 L) was added, and the solution was heated
to 40±458C. A seed of product was added followed by a
solution of oxalic acid .649 g, 7.21 mol, 0.5 equiv.) and
degassed MTBE .22.5 L) dropwise over 1.5±2 h at 40±
458C, which crystallized the product. The resulting colorless
slurry was stirred at 40±458C for 30 min followed by cool-
ing to 20±258C over 20 min. The suspension was stirred at
room temperature for 12 h, and the crystals isolated by
®ltration, washed with a 2:1 v/v MTBE/EtOH .17.72 kg,
9.40 kg) and dried under positive pressure of nitrogen
followed by in vacuo drying for 19 h at 408C to provide
3a .4.07 kg, 79%, 99.9 area% by HPLC) as colorless plates:
mp 128±1298C; 1H NMR.300.13 MHz, DMSO- d6) d 7.22±
7.34 .m, 10H), 5.95±6.35 .br, 4H exchangeable protons),
4.47 .s, 4H), 3.56 .s, 2H), 3.54 .s, 2H), 3.37 .m, 1H); 13C
NMR.75.47 MHz, DMSO- d6) d 164.9, 138.5, 128.7, 128.1,
128.0, 72.9, 68.1, 58.6. Anal. Calcd for C18H23N2O4: C,
65.24; H, 7.00; N, 8.45. Found: C, 65.17; H, 7.03; N, 8.43.
4.1.4. Preparation of hydrazone )11). 8.9% aqueous
NaOCl .titrated with aqueous Na2S2O3 prior to use;
18.4 kg, 22.0 mol) was added dropwise over 2 h to a stirred
mixture of 1,3-dibenzyloxy-2-propanol .9; 5.00 kg,
18.4 mol), 2,2,6,6-tetramethyl-1-piperidinyloxy free radical
.TEMPO; 287 g, 1.84 mol), MeCN .54.0 kg) and 3%
aqueous NaHCO3 .50.8 kg, 18.4 mol) in a 200-L glass
lined tank at 08C, keeping the internal temperature at 0±
58C. The resulting mixture was stirred for an additional
1 h at 0±58C and extracted with MTBE .100 kg) below
108C. The separated organic layer was washed with 10%
aqueous Na2SO3 .16.7 kg) below 108C followed by 5%
aqueous NaCl .10.5 kg) and 20% aqueous NaCl .15.0 kg)
at room temperature. The solution was then placed in the
200-L vessel and concentrated in vacuo .508C/60±
150 mmHg) to ca. 25 L and ¯ushed with n-heptane
.3£50 L) followed by dilution with n-heptane .68.2 kg).
The mixture was then warmed to 508C, and a solution of
Boc-NHNH2 .3.15 kg, 23.9 mol) in toluene .4.3 kg) was
added. The resulting mixture was stirred above 708C for

8922
A. Akao et al. / Tetrahedron 57 /2001) 8917±8923
4.1.6. Synthesis of penultimate intermediate )2). A 22-L
vessel was charged with DMA .8.3 L), anhydride 8
.1.00 kg, 0.94 mol) and hydrazine hemioxalate 3a .350 g,
1.06 mol) at 228C. The resulting slurry was degassed with
stirring by applying vacuum to the vessel .40±80 torr) for
5 min and ®lling with nitrogen .three cycles). The contents
were heated to 658C over 30 min during which time the
solution became homogeneous. Triethylamine .146 mL,
1.05 mol) was added rapidly and the solution aged at 658C
for 3 h. The contents were cooled to 458C and transferred to
a 50-L cylindrical vessel containing nitrogen-sparged
MTBE .17.0 L) held at 108C. The contents of the vessel
were again cooled to 108C and nitrogen-sparged water
.4.7 L) was added over 10 min to keep the internal tempera-
ture below 308C. 2N aqueous HCl .440 mL) was added to
the biphasic mixture at 228C. After agitation at 228C for
10 min, the layers were separated and the organic layer
washed with water .3£3.8 L). The organic layer was
concentrated in vacuo to the 5 L level .20±258C), and
multiple THF ¯ushes were performed to remove MTBE.
The solution concentration was adjusted to 175 g/L with
THF and utilized in the next reaction. Assay of the solution
indicated 1.25 kg of product ..99% yield). A portion of the
solution was evaporated to dryness to facilitate characteri-
lowered to 20% .w/v) by feeding i-PrOH .ca. 9 L) to the
vessel while keeping the batch volume at 7.5 L. The
contents were cooled to 708C and a seed of 1 .5.0 g) was
added as a slurry in i-PrOH .50 mL). The batch was held at
708C for 1 h followed by the addition of i-PrOH .5.0 L) over
90 min. The batch was aged at 708C for 9 h during which
time the bulk of the product crystallized. A constant volume
distillation feeding in i-PrOH .17 L) was performed that
resulted in lowering the water content to 3% .w/v). The
slurry was aged at 708C for 3 h .supernatant concen-
trationˆ5.3 g/L) followed by cooling to 228C and aging
for 1 h. The slurry was ®ltered and the cake was washed
with i-PrOH .2.5 L) and then methanol .1.5 L), followed by
drying in vacuo at 388C for 6 h to provide 1 .419 g, 82%,
99.4 area% by HPLC) as an orange solid: mp 3308C .dec);
[a]23 ˆ1117 .c 0.8, 1/1 MeCN/water); 1H NMR
D
.400.13 MHz, DMSO-d6-major rotamer) d 11.23 .s, 1H),
9.80 .s, 1H), 9.77 .s, 1H), 8.90 .d, Jˆ8.4 Hz, 1H), 8.82 .d,
Jˆ8.4 Hz, 1H), 7.21 .br s, 1H), 7.01 .br s, 1H), 6.84 .over-
lapping m, 2H), 6.00 .d, Jˆ8.0 Hz, 1H), 5.88 .t, Jˆ3.6 Hz,
1H), 5.57 .d, Jˆ2.4 Hz, 1H), 5.34 .d, Jˆ4.4 Hz, 1H), 5.13
.d, Jˆ4.4 Hz, 1H), 4.94 .d, Jˆ4.4 Hz, 1H), 4.56 .t, Jˆ
5.6 Hz, 2H), 4.04 .dd, Jˆ11.2, 3.2 Hz, 1H), 3.95 .over-
lapping m, 2H), 3.81 .dd, Jˆ10.4, 4.0 Hz, 1H), 3.53 .over-
lapping m, 6H); 13C NMR.100.64 MHz, DMSO- d6-major
rotamer) d 169.0, 168.9, 157.8, 157.6, 144.4, 143.1, 129.5,
127.9, 125.2 .2C), 118.9, 117.6, 115.9, 114.3, 114.2, 113.9,
110.3, 110.2, 97.5, 97.5, 84.5, 78.4, 76.8, 72.9, 67.5, 62.6,
60.5 .2C), 58.3.
1
zation. H NMR.600.13 MHz, DMSO- d6-selected data-
major rotamer) d 10.89 .s, 1H), 9.10 .d, Jˆ8.7 Hz, 1H),
9.01 .d, Jˆ8.7 Hz, 1H), 7.74 .d, Jˆ1.9 Hz, 1H), 6.99 .m,
1H), 6.83 .t, Jˆ7.6 Hz, 2H), 6.58 .d, Jˆ8.3 Hz, 1H), 6.13
.d, Jˆ7.6 Hz, 2H), 5.71 .d, Jˆ2.6 Hz, 1H), 5.31 .d, Jˆ
11.7 Hz, 1H), 5.26 .s, 2H), 5.06 .d, Jˆ11.7 Hz, 1H), 4.92
.d, Jˆ11.0 Hz, 1H), 4.81 .d, Jˆ13.2 Hz, 1H), 4.80 .s, 2H),
4.66, 4.64 .overlapping d's, 2H), 4.48 .d, Jˆ11.7 Hz, 2H),
4.41 .d, Jˆ11.7 Hz, 1H), 4.39 .d, Jˆ11.7 Hz, 1H), 4.29 .m,
2H), 4.09 .t, Jˆ8.7 Hz, 1H), 3.93 .d, Jˆ10.6 Hz, 1H), 3.86
.m, 2H), 3.81 .d, Jˆ10.0 Hz, 1H), 3.62 .m, 4H), 2.90 .d,
Jˆ10.0 Hz, 1H); 13C NMR.150.90 MHz, DMSO- d6-major
rotamer) d 168.6, 168.5, 159.0, 158.8, 143.3, 142.3, 138.1,
138.0, 138.0, 138.0, 137.2, 136.8, 136.8, 136.3, 129.3,
128.5, 128.4, 128.4, 128.4, 128.2, 127.9, 127.9, 127.9,
127.8, 127.8, 127.6, 127.6, 127.5, 127.4, 127.4, 127.2,
125.6, 125.5, 118.2, 117.7, 117.5, 115.8, 115.7, 115.4,
110.8, 110.3, 97.1, 96.2, 84.4, 82.8, 80.7, 76.0 .2C), 74.9,
74.3, 73.6, 72.8, 72.4, 72.3, 70.0, 70.0, 69.8, 69.5, 66.5,
57.5.
Acknowledgements
The authors would like to thank Drs Takashi Araki,
Catherine T. Pham, Vincent Antonucci, Michael Hicks
and Mr Keisuke Kamei for analytical support. Additional
technical support and valuable comments from Mr Ryosuke
Ushijima, Mr Masayuki Hagiwara and Mr Yoshiaki Kato
are also greatly acknowledged.
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