Synthesis of natural flutimide and analogous fully substituted pyrazine-2,6-diones, endonuclease inhibitors of influenza virus

Article abstract of DOI:10.1021/jo015665d

Flutimide, a fully substituted 1-hydroxy-3H-pyrazine-2,6-dione, is a fungal metabolite isolated from a new species of Delitschia cofertaspora. It has been shown to selectively inhibit cap-dependent endonuclease activity of influenza virus A. The inhibition of this activity is a target for the potential development of a therapeutic agent to treat influenza infections. A convergent total synthesis of flutimide starting from L-leucine has been described. The synthetic methodology has been extended to include the synthesis of specifically designed aromatic analogues of flutimide, some of which exhibited greater than 7-fold improvement in activity. The most potent compounds were those with p-fluorobenzylidene or p-methoxybenzylidene substitutions at C-5 of 3H-pyrazine-2,6-dione and showed IC₅₀ values of 0.9 and 0.8 μM, respectively. The details of the rationale for the synthetic design, syntheses, and biological activities of these analogues are described.

Full text of DOI:10.1021/jo015665d

5504
J . Org. Chem. 2001, 66, 5504-5516
Syn th esis of Na tu r a l F lu tim id e a n d An a logou s F u lly Su bstitu ted
P yr a zin e-2,6-d ion es, En d on u clea se In h ibitor s of In flu en za Vir u s
Sheo B. Singh*,† and J oanne E. Tomassini‡
Merck Research Laboratories, P.O. Box 2000, Rahway, New J ersey 07065, and Department of Antiviral
Research, Merck Research Laboratories, West Point, Pennsylvania 19486
sheo_singh@merck.com
Received April 4, 2001
Flutimide, a fully substituted 1-hydroxy-3H-pyrazine-2,6-dione, is a fungal metabolite isolated from
a new species of Delitschia cofertaspora. It has been shown to selectively inhibit cap-dependent
endonuclease activity of influenza virus A. The inhibition of this activity is a target for the potential
development of a therapeutic agent to treat influenza infections. A convergent total synthesis of
flutimide starting from L-leucine has been described. The synthetic methodology has been extended
to include the synthesis of specifically designed aromatic analogues of flutimide, some of which
exhibited greater than 7-fold improvement in activity. The most potent compounds were those with
p-fluorobenzylidene or p-methoxybenzylidene substitutions at C-5 of 3H-pyrazine-2,6-dione and
showed IC50 values of 0.9 and 0.8 µM, respectively. The details of the rationale for the synthetic
design, syntheses, and biological activities of these analogues are described.
Influenza is an acute and contagious respiratory
unique opportunity for such interventions.7 In 1994, we
reported7 a series of 4-substituted 2,4-dioxobutanoic acids
as specific inhibitors of the endonuclease activity of
transcriptase. Subsequently, we reported upon flutimide
(1a ), a novel fully substituted 3H-pyrazine-2,6-dione
isolated from the dung-inhabiting fungus Delitschia
confertaspora as a specific inhibitor of endonuclease.8
This compound exhibited an IC50 of 5.1 µM against
influenza virus transcriptase and had no activity against
other transcriptases (IC50 >150 µM). It inhibited cap-
dependent influenza endonuclease with an IC50 value of
6.8 µM. Although the structure of flutimide was deter-
mined by spectroscopic methods, confirmation by other
methods was needed due to structural ambiguities caused
by the presence of either heteroatoms or contiguous
quaternary carbons. Additionally, the yield of this com-
pound by microbial fermentation was very poor (<5 mg/
L), and an additional source of production was required
to provide an adequate supply for biological evaluations.
Therefore, total synthesis of flutimide was undertaken
and a convergent synthesis was designed to provide an
easy access to a variety of analogues.9 Details of the total
synthesis of flutimide (1a ), the synthesis of analogues
1-3, and their biological activities are described.
disease that is distinguished by a severe pulmonary
infection caused by influenza virus types A and B.1 Due
to periodic infections in humans of epidemic and pan-
demic proportions, influenza is a significant medical
problem. The epidemics caused by the airborne transmis-
sion of these viruses account for thousands of deaths in
the United States and millions of deaths worldwide every
year.2 Current therapies include a prophylactic annual
vaccination program and therapeutic treatments with
amantadine and rimantadine. More recently, neuramini-
dase inhibitors such as Relenza (zanamivir)3 and Tamiflu
(oseltamivir phosphate)4 have become available for treat-
ment, as well. However, these approaches have limited
utility, and there is a significant demand for development
of additional anti-influenza vaccines and therapeutic
agents with potentially different mechanisms of action.2a,5
There are numerous potential molecular targets that
can be considered for therapeutic intervention for treat-
ment of influenza infections.6 Primary transcription and
inhibition of influenza specific endonuclease represent a
* To whom correspondence should be addressed. Tel: (732) 594-
3222. Fax: (732) 594-6880. E-mail: sheo_singh@merck.com.
† Merck Research Laboratories, Rahway, NJ .
‡ Merck Research Laboratories, West Point, PA.
(1) (a) Kingsbury, D. W. In Virology, 2nd ed.; Fields, B. N., Knipe,
D. M., Eds.; Paven Press Ltd., New York, 1990; p 1075. (b) Plotch, S.
J .; Bouloy, M.; Ulmanen, I.; Krug, R. M. Cell 1981, 23, 847. (c) Palese,
P.; Ritchey, M. B.; Schulman, J . L. J . Virol. 1977, 21, 1187.
(2) (a) Douglas, R. G., J r. New Engl. J . Med. 1990, 322, 443. (b)
Kilbourne, E. D. Influenza, Plenum Press: New York, 1987.
(3) (a) Zanamivir: Ann. Rep. Med. Chem. 1999, 35, 352. (b) For a
review, see: McKimm-Breschkin, J . L. Antiviral Res. 2000, 47, 1.
(4) (a) Oseltamivir phosphate: Ann. Rep. Med. Chem. 1999, 35, 346.
(b) See ref 3b.
(5) Hayden, F. G.; Hay, A. J . Curr. Top. Microbiol. Immunol. 1992,
176, 1304.
(6) For reviews on various influenza targets and development of
inhibitors, see: (a) Meanwell, N. A.; Krystal, M. Drug Discovery Today
1996, 1, 316. (b) Meanwell, N. A.; Krystal, M. Drug Discovery Today
1996, 1, 388. For resistance, see: (c) McKimm-Breschkin, J . L.
Antiviral Res. 2000, 47, 1.
10.1021/jo015665d CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/07/2001

Synthesis of Natural Flutimide
J . Org. Chem., Vol. 66, No. 16, 2001 5505
Sch em e 1
envisioned for the key intermediate 6a . The first, less
flexible but cleaner than the second route, could be
envisioned via intermediate 7a by the N-alkylation of an
R-amino acid ester (8a , P2 ) alkyl) with an R-halo-alkyl
acetate (9). Alternatively, 6a could be prepared by mono
C-alkylation of a symmetrical cyclic intermediate 6b that
could be prepared by analogous N-alkylation of glycine
ester (8b, P2 ) alkyl) with an R-halo-alkyl acetate. The
cyclic intermediate 6b provides additional flexibility for
introduction of various alkyl groups from alkyl halides
at a more advanced stage. However, the first route was
favored for the synthesis of 6a due to the simplicity. This
route allowed for the possibility of performing multiple
steps of reactions without chromatography. In both
routes, the introduction of an N-hydroxy group was
envisaged to employ hydroxylamine.12 A p-methoxybenzyl
(PMB) group was selected for protection of the amino
group that could be oxidatively cleaved with DDQ or CAN
and would be expected to also oxidize the amine to the
∆3-imine in situ. A MOM group was selected for the
protection of the N-hydroxy group, which later could be
cleaved by TFA. The synthesis based on this approach is
described below.
Syn th esis of Mon osu bstitu ted P ip er a zin e-2,6-d i-
on es. The N-alkylation of (S)-Leu-OMe.HCl (10a , R )
i-Pr) or (S)-Phe-OMe (10b, R ) Ph) at 60 °C with
equimolar amounts of R-bromo-tert-butyl acetate in CH3-
CN in the presence of diisopropylethylamine (DIPEA)
gave the corresponding N-alkylated derivative 11 (Scheme
2). After completion of the reaction, the product was
heated with p-methoxybenzyl (PMB) chloride in the same
reaction vessel to afford compound 12 in a 90-97%
overall two-step yield.13 Treatment of the ester (12) with
TFA selectively hydrolyzed the tert-butyl ester and
produced free acid 13 in 94% yield. The acid group of 13
was activated with N-hydroxysuccinimide to produce
active ester 14, which was then reacted with neutralized
hydroxylamine to give compound 15, and this was
subsequently heated at reflux to afford the cyclized
product 16. The N-hydroxy group of 16 was conveniently
reacted with MOM chloride to afford MOM ether 17 in
>80% purified yield based on acid 13. This entire reaction
scheme was conveniently carried out on a >0.25 mol scale
without need for any purification until the last step of
the Scheme 2.
Resu lts a n d Discu ssion
Retr osyn th etic An a lysis. Flutimide (1a ) is a fully
substituted 1-hydroxy-3H-pyrazine-2,6-dione and con-
tains the following sensitive groups: an N-hydroxyimide,
an imine, and an exocyclic enamine with Z-geometry. The
endocyclic imine may help in the stabilization of the
exocyclic enamine via extended conjugation. The combi-
nation of such structural features is unusual and un-
precedented.10 Thus, there were two goals for the
synthesis of flutimide (1a ). The first goal was to confirm
the proposed structure of natural flutimide by total
synthesis, and the second goal was to generate analogues
to develop a structure-activity relationship. A prereq-
uisite of the synthetic design was that it must be
convergent and provide maximal flexibility for the syn-
thesis of analogues. The retrosynthetic analysis is shown
in Scheme 1, which entailed four key operations: (i) the
selection of a protecting group for the N-hydroxy group
that could be cleaved with TFA,11 (ii) selection of an
N-protecting group that could be cleaved oxidatively and
could concomitantly generate the required endocyclic
imine, (iii) a cyclization step to yield a piperazine-2,6-
dione either with or without one of the side chains (R1),
and (iv) easy installation of the exocyclic olefin on the
piperazine-2,6-dione with various R.
In this strategy, deprotection of the enamine-installed
advanced intermediate 4 provides the desired product,
while 4 could be derived from putative intermediate 5
by â-elimination of a hydroxy group that could in turn
be easily prepared by aldol condensation of cyclic inter-
mediate 6a with diverse aldehydes. Two routes could be
Ad d ition of th e Left Sid e Ch a in s. Aldol condensa-
tion of 17a with isobutyraldehyde and lithium hexa-
methyldisilazide (LHMDS) at -78 °C exclusively pro-
duced the 3S,5S,11R-stereoisomer of flutimide advanced
hydroxy intermediate 18a in 63% yield on a 5 g scale
(Scheme 3).
(7) (a) Tomassini, J .; Selnick, H.; Davies, M. E.; Armstrong, M. E.;
Baldwin, J .; Bourgeois, M.; Hasting, J .; Hazuda, D.; Lewis, J .;
McClements, W.; Ponticello, G.; Radzilowski, E.; Baldwin, J .; Bour-
geois, M.; Ponticello, G.; Smith, G.; Tebben, A.; Wolfe, A. Antimicrob.
Agents Chemother. 1994, 38, 2827. (b) Hastings, J .; Selnick, H.;
Wolanski, B.; Tomassini, Antimicrob. Agents Chemother. 1996, 38,
1304.
(8) (a) Hensens, O. D.; Goetz, M. A.; Liesch, J . M.; Zink, D. L.; Helms,
G. L.; Singh, S. B. Tetrahedron Lett. 1995, 36, 2005. (b) Tomassini, J .
E.; Davies, M. E.; Hastings, J . C.; Lingham, R.; Mojena, M.; Raghoobar,
S. L.; Singh, S. B.; Tkacz, J . S.; Goetz, M. A. Antimicrob. Agents
Chemother. 1996, 40, 1189.
The stereochemistry and conformation of 18a was
deduced by NOE difference spectroscopy in C6D6. Selected
NOE enhancements are shown in Figure 1. Irradiation
of the C-7 methylene protons (δ 1.40) produced strong
enhancements to H-5, indicating the 1,3-diaxial relation-
ship between the (S)-leucine derived side chain and H-5,
consequently establishing the S stereochemistry at C-5.
(12) It would be better if the N-hydroxy group could be directly
introduced as a last step, but there are no methods available which
will allow N-hydroxylation in good yield. We investigated MoO5‚HMPA
oxidation20 on 2,5-piperazinedione,8a which gave very poor yield.
Therefore, we did not investigate this oxidation method in this project
and decided to introduce the N-hydroxy group from hydroxylamine.
(13) Both alkylations could be effected in ethanol but CH3CN proved
to be much better and gave cleaner products in the large-scale
reactions.
(9) For a brief account of the synthesis, see: Singh, S. B. Tetrahedron
Lett. 1995, 36, 2009.
(10) At the time of the discovery of flutimide, a computer based CAS
search of 3-alkyl-3H-pyrazine-2,6-dione showed no hits and did not
change significantly when the search was performed again in J anuary
2001 except for the report on flutimide8 and analogues18 and one other
report of pyrazinecarboxamide.19
(11) Flutimide had previously been shown to be stable to TFA.8a

5506 J . Org. Chem., Vol. 66, No. 16, 2001
Singh and Tomassini
Sch em e 2
NCH2 protons (H-15, δ 3.55, 1H) along with H-5, H-12,
and CH3-13 (δ 1.00), indicating R-stereochemistry at
C-11, which was further supported by formation of
predominantly the Z-olefin as the elimination product
(vide infra). The irradiation of the second NCH2 proton
(δ 3.19, 1H) gave strong enhancements to H-3 and only
weak enhancements to H-5. Unfortunately, the NOE
effect of the NCH2 (δ 3.55) to either H-3 or H-5 could not
be unambiguously discerned due to their proximal chemi-
cal shifts.
Similar aldol condensation reactions of 17a with benz-
aldehyde, p-methoxybenzaldehyde, and p- and m-fluoro-
benzaldehyde exclusively furnished the corresponding
3S,5S,11R-hydroxy products 18b-e. These aldol reac-
tions were highly diastereoselective, just like 18a , and
did not produce any of the other three potential diastereo-
mers. However, the aldol reaction of 17a with the
hindered o-flurorobenzaldehyde gave an approximately
2.5:1 mixture14 of 3S,5S,11R and 3S,5S,11S isomers 18f
and 19f, respectively, in rather poor combined yield
(33%). The aldol reaction of phenylalanine-derived
piperazine-2,6-dione 17b with isobutyraldehyde surpris-
ingly also gave a 2:1 diastereomeric mixture14 of 18g and
19g, whereas the analogous aldol reactions of 17b with
p-methoxybenzaldehyde and p-fluorobenzaldehyde af-
forded only 18h and 18i, respectively.
F igu r e 1. Structure of 18a showing selected NOE enhance-
ments in C6D6.
Sch em e 3
The C-5 stereospecificity of the aldol reaction appears
to be controlled by the stereochemistry at C-3, presum-
ably due to the axial orientation of the C-3 side chain.
This allows substitution at C-5 only from the equatorial
side, resulting in the S-isomer. Stereospecificity at C-11
must result from the steric bulk of the PMB group.
However, the steric bulk present near the aldehyde group
appears to compete (o-fluorobenzaldehyde vs other benz-
The newly generated hydroxy group (δ 3.30, brs) at C-11
appears to form a hydrogen bond with the C-6 carbonyl
oxygen and tends to fix the conformation of H-11 syn to
H-5 (J ) 9.5 Hz). Irradiation of H-11 exhibited unam-
biguous strong NOE enhancements to one of the two
(14) No attempts were made to optimize this reaction either for the
diastereoselectivity or for yield.

Synthesis of Natural Flutimide
J . Org. Chem., Vol. 66, No. 16, 2001 5507
Sch em e 4
Sch em e 5
Sch em e 6
gave the expected oxidized product 23a in ∼30% yield
after chromatography (Scheme 5).15 Similar oxidations
of 21b-i gave corresponding oxidized product 23b-i in
19-50% yields. The low yields of these reactions were
attributed to the decomposition of the product, presum-
ably caused by hydration and subsequent cascade of
rearrangement reactions. The oxidation reactions of 21c
and 21g produced significant amounts of the correspond-
ing R-ketoamides 24 and 25 along with the expected
oxidized products 23c and 23g, respectively. Although
most of these reactions produced some amounts of either
24 or 25, the latter reaction produced equal proportions
of R-ketoamide 25 and 23g, indicating that the aromatic
group facilitated the formation of R-ketoamides. Many
of these oxidations also produced the corresponding
aldehydes, presumably after addition of water followed
by retro-aldol reactions. These side reactions were col-
lectively responsible for the lower yields in the oxidation
reactions.
F igu r e 2. Structure of 21a showing selected NOE enhance-
ments in CDCl3.
aldehydes) for the stereoselection and disturbs the stereo-
selectivity at C-11.
â-Elim in a tion Rea ction . The aldol product 18a was
reacted with methanesulfonyl chloride (MsCl), DIPEA,
and DMAP at -23 °C to give in quantitative yield the
corresponding mesylate 20a , which was directly used for
the elimination reaction. The â-elimination reactions
turned out to be challenging and required optimization.
Thus, the reaction of mesylate at 0 °C with 3 equiv of
DBU in toluene gave 74% combined yield of a 3:1 Z/E
mixture of olefins 21a and 22a , respectively (Scheme 4).
The olefin geometries of 21a and 22a were deduced
by NOE difference spectroscopic measurements of 21a
and are shown in Figure 2. Irradiation of the aromatic
doublet (δ 7.20, J ) 8.8 Hz) of 21a exhibited strong
enhancements to the vinylic methine proton (δ 3.11),
which in turn showed enhancements to the NCH2 protons
(δ 3.81) indicating a Z-geometry of the olefin. This
assignment was further supported by a ∆ 1.27 ppm
downfield shift of the olefinic proton of 21a due to the
oxygen proximity effect of C-6 ketone compared to the
isomeric E-olefin 22a .8a The methine protons of both
isopropyl groups showed NOE enhancements to each
other, indicating the spacial proximity of these protons
due to their freedom of rotations.
The oxidation of the E-isomer 22a did not give any of
the expected products but instead produced a pair of
isomeric16 imidazolinediones 26 and 27, as well as
R-ketoamide 24 (Scheme 6).
Although there was decomposition observed during
oxidation of Z-olefins, none of the reactions produced
products analogous to compound 26 and 27. It is surpris-
(15) The oxidation reaction with ceric ammonium nitrate (CAN)
caused decomposition and was not successful.
(16) The geometry of the olefins was deduced by the comparison of
1H NMR spectra of compounds 26 and 27 and application of oxygen
proximity induced effect on the 1H chemical shifts. The Z-olefinic proton
(δ 5.73) of 26 showed
∆ 0.5 ppm downfield shift compared to
Similar reactions of aromatic alcohols 18b-f and
18h -i with MsCl gave corresponding mesylates 20b-f,
and 20h -i, which spontaneously â-eliminated to produce
corresponding Z-olefins 21b-f and 21h -i in 70% to 99%
overall yields. The elimination reaction of the mixture
of 18g and 19g produced a mixture of Z/E isomers 21g
and 22b.
corresponding E-olefinic proton (δ 5.28) of 27 due to proximity to C-6
keto oxygen.8a This is supported by the observation of an analogous
but more pronounced ∆ 1 ppm downfield shift of the vinylic methine
proton of the isopropyl group of the E-isomer 27 (δ 3.66) compared to
corresponding proton of Z-isomer 26 (δ 2.64) due to the same effect.
(17) The details of the assays and the biological activity of some of
the compounds have been previously reported in ref 8b.
(18) Singh, S. B. US Patent 5,624,928 (April 29, 1997).
(19) Furuta, Y.; Egawa, H. PCT Int. Appl. WO2000010569 A1
20000302, 1999.
Oxid a tion a n d Dep r otection Rea ction s. The oxida-
tion of 21a with DDQ in a mixture of CH2Cl2-H2O (2:1)
(20) Rigby, J . H.; Qabar, M. J . Org. Chem. 1989, 54, 5852.

5508 J . Org. Chem., Vol. 66, No. 16, 2001
Singh and Tomassini
F igu r e 3. Plausible mechanism of formation of unexpected products.
Sch em e 7
ing that a mere difference in the olefinic geometry caused
such a dramatic difference in the product composition.
The process of this decomposition and formation of
compounds 26 and 27 is pronounced, presumably due to
decreased steric factors emanating from E-olefin. A
plausible mechanism for the formation of the undesired
products 24-27 is shown in Figure 3. There are two
potential initial routes for the formation of isomeric
compounds 26 and 27 (upper panel). However, the
formation of compound 24 could be explained by a
mechanism that involves the oxidation of the amine
followed by addition of water and rearrangements (lower
panel).
Sch em e 8
The MOM ethers 23a -i were reacted with TFA in
CH2Cl2 to give the corresponding N-hydroxy compounds
1-3 in 15-86% yields. These compounds were purified
either by chromatography on reversed-phase HPLC or
by recrystallization. The reaction yield was very good,
but we observed a significant loss of material during
chromatography. Small-scale (<10 mg) purification of
synthetic flutimide could be easily accomplished by
reversed-phase HPLC, but injection of larger quantities
of material resulted in extremely low recoveries. Most of
the aromatic pyrazinediones were crystalline and were
purified by recrystallization, resulting in much better
yield of the products. The NMR, MS, HPLC, and biologi-
cal properties of synthetic compound 1a were indistin-
guishable from the natural flutimide.
The deprotection of 23i with TFA produced a 1:1
isomeric mixture of 2b and 3 as a result of enamine-
imine tautomerization. Surprisingly, the aromatic ana-
logue 2a did not produce any tautomeric products. TFA
treatment of 23b produced expected product 2c, which
spontaneously tautomerized and produced conjugated
product 1b, which was also produced by a similar reaction
of 23b.
MOM ether with methanolic sulfuric acid gave 29, which
was reacted with titanium trichloride to give N-deoxy
compound 30 in moderate yield. Compound 30 was
reacted with methanesulfonyl chloride to give the
mesylate derivative which concomitantly â-eliminated to
give 63% yield of enamine 31. This was oxidized with
DDQ, giving 36% yield of pyrazine-2,6-dione 32 (Scheme
8).
Tetr a h yd r o- a n d N-Deoxy Der iva tives. To test the
role of the enamine-imine or N-hydroxy group on the
transcriptase activity, compounds 28 (Scheme 7) and 32
(Scheme 8) were synthesized. Hydrogenation of com-
pound 2b produced tetrahydro compound 28 without
reductive elimination of the N-hydroxy group.
For synthesis of the N-deoxy compound with an intact
enamine-amine group, we elected to synthesize the
corresponding deoxy analogue 32 of the most active
p-fluorobenzylidene analogue 1d and began the synthesis
from the advanced intermediate 29. Deprotection of
Biologica l Activity. Flutimide (1a ) inhibited cap1
ALMV primed influenza transcriptase with an IC50 value
of 5.1 µM.8b There was no difference in the activities of
the natural and synthetic samples of flutimide (1a ). The
substitution of the left side isopropyl group of flutimide
with p-substituted phenyl groups (1c and 1d ) produced
some of the most active compounds of the series and
yielded IC50 values of 0.8 and 0.9 µM, respectively (Table
1).17 Surprisingly, there was no significant difference in
the activities between p-methoxyphenyl (1c) and p-
fluorophenyl (1d ) substituted compounds. However, there

Synthesis of Natural Flutimide
J . Org. Chem., Vol. 66, No. 16, 2001 5509
Ta ble 1. ALMV P r im ed Tr a n scr ip ta se In h ibitor y
Activities of Com p ou n d s
(2 × 100 mL), 10% aqueous citric acid (100 mL), and water
(100 mL). The EtOAc extract was dried over Na2SO4, evapo-
rated under reduced pressure, and chromatographed over
silica gel column. Elution with 5% EtOAc in hexane gave pure
N-tert-butyl acetyl -(S)-leucine methyl ester (11a ) as colorless
compd
IC50 (µM)
compd
IC50 (µM)
1a
1b
1c
1d
1e
1f
5.1
4.8
0.8
0.9
1.5
7.3
2a
2b
3
18-22
23
2.8
3.5
6.5
oil: [R]25 -17.2° (c 2.9, MeOH); 1H NMR (CDCl3) δ 0.88 (3H,
D
d, J ) 6.6 Hz), 0.89 (3H, d, J ) 6.6 Hz), 1.42 (9H, s), 1.48 (2H,
m), 1.70 (1H, apparent hept, J ) 6.6 Hz), 3.24 (2H, ABq, J )
18 Hz), 3.29 (1H, t, J ) 7.2 Hz), 3.68 (3H, s); 13C NMR (CDCl3)
δ 22.3, 22.7, 24.8, 28.1, 42.4, 49.9, 51.7, 59.2, 81.3, 170.9, 175.4.
Anal. Calcd for C13H25NO4: C, 60.20; H, 9.72; N, 5.40. Found:
C, 60.05; H, 9.44; N, 5.32.
>200
>200
85
28
32
>365
was a marked difference in the activity when compared
with phenyl (1b), m- (1e), and o-fluorophenyl (1f) ana-
logues. Substitution of both of the isopropyl groups with
phenyl and/or p-methoxyphenyl (2a ), p-fluorophenyl (2b),
and 3 produced compounds with activity slightly greater
than or equivalent to that of flutimide, but not as potent
as 1c. The reduction of olefins (compound 28) caused a
17-fold loss of activity, and the reduction of the N-hydroxy
group (compound 32) completely obliterated the tran-
scriptase activity. All other pyrazine-2,6-diones (23) and
piperazine-2,6-diones (18-22) were inactive (>200 µM).
Flutimide-inhibited influenza virus infection of MDCK
cells with an IC50 of 5.9 µM without any toxicity at 100
µM concentrations. The more potent in vitro active
compounds 1c and 1d also appeared to be more potent
in the antiviral assay but showed overt cytotoxicity to
the cells at >10 µΜ concentrations. The SAR indicated
that the substitution of both side chains was tolerated
and produced 5-6-fold more potent compounds. This
suggests that the preparation of further derivatives can
potentially lead to superior analogues; however, the
N-hydroxy group and both olefins appear to be necessary
for the potency.
p-Methoxybenzyl chloride (56 mL, 0.41 mol) was added to
the aforementioned reaction mixture, which was then heated
at 70 °C for 24 h. After completion of the reaction, the solution
was allowed to cool to room temperature. CH3CN and DIPEA
were removed under reduced pressure, and the residue was
suspended in 500 mL of water. The product was extracted with
EtOAc (3 × 600 mL). The EtOAc extract was sequentially
washed with water (2 × 400 mL), 10% aqueous citric acid (400
mL), and water (400 mL) and dried (Na2SO4). The EtOAc was
removed under reduced pressure, and the product was chro-
matographed over silica gel and eluted with 5% EtOAc-
hexane to yield colorless oil of N-p-methoxybenzyl-N-tert-
butylacetyl-(S)-leucine methyl ester 12a (101.5 g, 97.3%):
1
[R]25 -67.8° (c 3.43, MeOH); H NMR (CDCl3) δ 0.73 (3H, d,
D
J ) 6.6 Hz), 0.86 (3H, d, J ) 6.6 Hz), 1.43 (9H, s), 1.48 and
1.57 (2H, m), 1.76 (1H, apparent hept, J ) 6.6 Hz), 3.34 (2H,
ABq, J ) 17.4 Hz), 3.42 (1H, t, J ) 6.6 Hz), 3.66 (1H, ABd, J
) 13.5 Hz), 3.69 (3H, s), 3.78 (3H, s), 3.90 (1H, ABd, J ) 13.5
Hz), 6.83 (2H, d, J ) 8.7 Hz), 7.30 (2H, d, J ) 8.7 Hz); 13C
NMR (CDCl3) δ 21.9, 23.1, 24.3, 28.1, 39.2, 51.14, 52.4, 55.3,
55.5, 60.5, 80.5, 113.6, 130.2, 131.0, 158.8, 171.1, 173.9. Anal.
Calcd for C21H33NO5: C, 66.46; H, 8.76; N, 3.69. Found: C,
66.75; H, 8.93; N, 3.64.
Syn th esis of N-p-Meth oxyben zyl-N-ter t-bu tyla cetyl-
(S)-p h en yla la n in e Meth yl Ester (12b). (S)-Phenylalanine
methyl ester hydrochloride (20 g, 92.7 mmol) was reacted with
R-bromo-tert-butyl acetate (16.07 mL, 98 mmol) in CH3CN for
6 h before addition of p-methoxybenzyl chloride (18.85 mL, 139
mmol) and then heated at 70 °C overnight. The reaction was
worked up and chromatographed as described above to give
pure N-p-methoxybenzyl-N-tert-butyl acetyl-(S)-leucine methyl
Su m m a r y
In this paper, we have described the convergent total
synthesis of natural flutimide, which was successfully
extended to the synthesis of additional analogues with
substitutions at positions 1, 3, and 5 of 3H-pyrazine-2,6-
dione. The p-substituted phenyl-substituted analogues
showed activity (IC50) in sub µM levels that are 6-7-fold
better than flutimide.
ester 12b (34.0 g, 89.1%): [R]25 -52.8° (c 1.88, MeOH); 1H
D
NMR (CDCl3) δ 1.44 (9H, s), 2.95 (1H, dd, J ) 13.5, 7.2 Hz),
3.04 (1H, dd, J ) 13.8, 8.1 Hz), 3.40 (2H, ABq, J ) 17.4 Hz),
3.64 (3H, s), 3.64 (1H, dd, J ) 7.2, 8.1 Hz), 3.74 (1H, d, J )
13.5 Hz), 3.78 (3H, s), 3.94 (1H, d, J ) 13.5 Hz), 6.76 (2H, d,
J ) 9.0 Hz), 7.10 (2H, d, J ) 8.7 Hz), 7.11 (2H, dd, J ) 8.1,
1.5 Hz), 7.22 (3H, m); 13C NMR (CDCl3) δ 28.1, 36.4, 51.3, 52.2,
55.2, 55.4, 64.3, 80.8, 113.5, 126.3, 128.2, 129.4, 130.0, 130.6,
138.0, 158.7, 170.8, 172.7; HRESI-FTMS calcd for C24H32NO5
Exp er im en ta l Section
Gen er a l P r oced u r e. All reagents were purchased from
Sigma-Aldrich Co. and were used without further purification.
Optical rotations were measured on a Perkin-Elmer model 241
polarimeter. All NMR spectra were recorded on Varian Inova
instruments operating at 400 and 500 MHz for 1H and 100
and125 MHz for 13C nuclei or on a Varian XL300 operating at
300 MHz for 1H and 75 MHz for 13C nuclei. FAB-MS was
performed on J EOL instrument. LC-MS was performed on a
Thermo Quest LCQ instrument using electrospray ionization
(ESI) or atmospheric pressure chemical ionization (APCI).
High-resolution mass spectral analysis was performed on a
Thermo Quest FTMS using electrospray ionization. LCMS and
1H NMR spectral analysis was used to determine the purity
of all compounds.
Syn th esis of N-p-Meth oxyben zyl-N-ter t-bu tyla cetyl-
(S)-leu cin e Meth yl Ester (12a ). A solution of R-bromo-tert-
butyl acetate (44.4 mL, 0.275 mol), (S)-leucine methyl ester
hydrochloride (50 g, 0.275 mol), and DIPEA (191 mL, 1.1 mol)
in CH3CN (400 mL) was stirred at room temperature for 16 h
and heated at 60 °C for 4 h. After completion of the reaction
(TLC, hexanes-EtOAc, 9:1), a small aliquot (5 mL) was taken
out and CH3CN was removed under reduced pressure. EtOAc
(100 mL) was added, and the solution was washed with water
(M + H) 414.2280, found 414.2263. Anal. Calcd for C24H31-
NO5: C, 69.71; H, 7.56; N, 3.39. Found: C, 69.79; H, 7.49; N,
3.16.
Syn th esis of N-p-Meth oxyben zyl-N-ca r boxym eth yl-
(S)-leu cin e Meth yl Ester (13a ). To a solution of tert-butyl
ester (12a , 114 g, 0.3 mol) in methylene chloride (200 mL) at
-78 °C was added trifluoroacetic acid (160 mL). After the
solution was stirred for 20 min at -78 °C, it was allowed to
warm to room temperature and stirred for 70 h. Volatile
material was removed under reduced pressure. EtOAc (2.0 L)
was added to the residue followed by 100 mL of water. The
pH of the solution was 1.0, which was basified to pH 4.0 by
addition of 10% aqueous solution of NaHCO3. The layers were
separated, and the EtOAc solution was washed once with 400
mL of water. The product was extracted from EtOAc solution
by extraction with 10% aqueous NaHCO3 (3 × 300 mL), 4 N
NaOH (400 mL), and water (200 mL). The EtOAc extract was
dried over Na2SO4 and concentrated to give unreacted starting
material (30 g). All of the basic extracts containing the product
were combined and acidified to pH 4.0 by addition of aqueous
citric acid and extracted with EtOAc (3 × 500 mL). The
combined EtOAc extract was washed with water (400 mL),
dried over Na2SO4, and evaporated under reduced pressure

5510 J . Org. Chem., Vol. 66, No. 16, 2001
Singh and Tomassini
followed by drying at 60 °C under vacuum for 48 h to give
pure acid 13a (67 g, 93.7%, based on recovery of the starting
material) as a gum. A small aliquot of the acid was purified
on a preparative Zorbax RX C-8 (22.5 × 250 mm) HPLC
column and eluted with 30% aqueous CH3CN at a flow rate of
58.5, 61.4, 114.1, 128.1, 130.3, 159.4, 165.4, 169.0; FABMS m/z
367 (M + Na + K - H), 351 (M + 2Na - H), 336 (M + Na +
Li - H), 319 (M + 2Li - H). Anal. Calcd for C16H22N2O4‚0.3
TFA: C, 58.54; H, 6.60; N, 8.23. Found: C, 58.85; H, 6.51; N,
8.50.
10 mL/min to give an analytical sample: [R]25 -33.8 (c 0.32,
To a cooled (-40 °C) and stirred (under nitrogen) solution
of just prepared N-hydroxy compound 16a (66.8 g, 0.2 mol) in
methylene chloride (600 mL) was added DIPEA (76 mL, 0.4
mol) followed by addition of methoxymethyl chloride (33.1 mL,
0.4 mol) over 15 min. The solution was stirred at the same
temperature for 2 h. After completion (TLC, hexanes-EtOAc,
9:1) of the reaction, methylene chloride was removed under
reduced pressure, water (500 mL) was added to the residue,
and product was extracted with EtOAc (3 × 600 mL). The
EtOAc layer was washed with 10% aqueous citric acid (2 ×
400 mL) and water (2 × 400 mL), dried over Na2SO4, and
evaporated under reduced pressure to give ∼80 g of an oily
product that was chromatographed on a silica gel column.
Elution with 5-10% of EtOAc in hexane gave MOM ether 17a
D
MeOH); 1H NMR (CDCl3) δ 0.85 (3H, d, J ) 6.3 Hz), 0.90 (3H,
d, J ) 6.0 Hz), 1.68 (2H, m), 1.83 (1H, m), 3.81 (3H, s), 3.82
(3H, s), 3.96 (2H, ABq, J ) 17.4), 4.05 (1H, dd, J ) 8.7, 4.8
Hz), 4.27 (2H, s), 6.92 (2H, d, J ) 8.7 Hz), 7.33 (2H, d, J ) 8.7
Hz); FABMS m/z 330 (M + Li), 324 (M + H). Anal. Calcd for
C
17H25NO5.0.7 TFA: C, 54.81; H, 6.42; N, 3.49. Found: C,
54.89; H, 6.25; N, 3.83.
Syn th esis of N-p-Meth oxyben zyl-N-ca r boxym eth yl-
(S)-p h en yla la n in e Meth yl Ester (13b). tert-Butyl ester
(12b, 33 g, 80 mmol) in methylene chloride (160 mL) was
deprotected with trifluoroacetic acid (40 mL) by stirring at
room temperature for 48 h. The solvents were removed under
reduced pressure, the product was thoroughly dried under
vacuum for 48 h, and the acid (13b) was used for next step
without any purification: 1H NMR (CDCl3) δ 3.24 (1H, dd, J
) 13.5, 8.1 Hz), 3.34 (1H, dd, J ) 13.5, 7.5 Hz), 3.74 (3H, s),
3.79 (3H, s), 4.14 (2H, s), 4.34 (2H, ABq, J ) 12.9 Hz), 4.40
(1H, dd, J ) 8.1, 7.5 Hz), 6.86 (2H, d, J ) 8.7 Hz), 7.07 (2H,
m), 7.18 (2H, d, J ) 9.0 Hz), 7.31 (3H, m); HRESI-FTMS calcd
for C20H24NO5 (M + H) 358.1649, found 358.1634.
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyben zylp ip er a zin e-2,6-d ion e (17a ). N-Hydroxysuccinim-
ide (23.0 g, 0.2 mol) and TEA (27.8 mL, 0.2 mol) was added to
a soultion of acid 13a (64.6 g, 0.2 mol) in methylene chloride
(500 mL). The solution was cooled to 0 °C, and DCC (41.2 g,
0.2 mol) was added in small portions over a period of 5 min.
The solution was stirred under nitrogen for 20 h at room
temperature. The precipitated urea was removed by filtration,
and the filtrate was concentrated to dryness under reduced
pressure to give 90 g of chromatographically homogeneous
(TLC, hexanes-EtOAc, 7:3) succinimide ester (14a ) as a gum
that was used in the next step without purification: 1H NMR
(CDCl3) δ 0.70 (3H, d, J ) 6.6 Hz), 0.86 (3H, d, J ) 6.6 Hz),
1.54 and 1.61 (2H, m), 1.73 (1H, m), 2.84 (4H, s), 3.46 (1H, dd,
J ) 9.0, 6.3 Hz), 3.69 (1H, d, J ) 12.9 Hz), 3.71 (3H, s), 3.79
(3H, s), 3.86 (2H, ABq, J ) 18 Hz), 3.94 (1H, d, J ) 13.2 Hz),
6.84 (2H, d, J ) 8.7 Hz), 7.28 (2H, d, J ) 8.7 Hz); FABMS m/z
421 (M + H).
To a solution of succinimide ester 14a (84 g, 0.2 mol) in a
mixture of ethanol (250 mL) and THF (200 mL) was added a
neutralized solution of hydroxylamine [hydroxylamine hydro-
chloride (20.85 g, 0.3 mol) was dissolved in 100 mL of water
and was mixed with a 100 mL solution of 0.3 mol NaOH]. The
mixture, which had a pH of 6.0, was stirred at room temper-
ature for 3 h. TLC examination (hexanes-EtOAc, 7:3) indi-
cated consumption of the succinimide ester and formation of
an FeCl3 positive product 15a . Precipitated NaCl was removed
from the reaction mixture by filtration, and the precipitate was
washed with 20 mL of ethanol. The combined filtrate was
refluxed overnight to give almost clean (HPLC, Zorbax RX C-8,
4.6 × 250 mm, 40% aqueous CH3CN containing 0.1% TFA,
flow rate 1 mL/min at 40 °C, tR of the product 15.19 min)
cyclized N-hydroxy compound (16a ). After removal (under
reduced pressure) of most of the solvents from the reaction
mixture (pH 5.3), it was poured onto 200 mL water giving a
total volume of 600 mL, which was extracted with EtOAc (4
× 900 mL). The EtOAc extract was washed with water (300
mL), dried over Na2SO4, and evaporated under reduced
pressure to give ∼70 g of crude product as a gum. A small
aliquot of the product was purified on preparative Zorbax RX
C-8 (22.5 × 250 mm) HPLC column and eluted with 30%
aqueous CH3CN containing 0.1% TFA at a flow rate of 10 mL
per min followed by lyophilization of the fractions gave 16a
as a semisolid: [R]25D -21.2° (c 0.61, CH3OH); 1H NMR (CDCl3)
δ 0.80 (3H, d, J ) 6.3 Hz), 0.94 (3H, d, J ) 6.3 Hz), 1.28 (2H,
m) 1.60 (1H,m), 3.35 (1H, brt), 3.58 (1H, d, J ) 18 Hz), 3.72
(2H, ABq, J ) 12.9 Hz), 3.80 (3H, s), 3.87 (1H, d, J ) 18 Hz),
5.90 (1H, br, OH), 6.86 (2H, d, J ) 8.7 Hz), 7.17 (2H, d, J )
8.4 Hz); 13C NMR (CDCl3) δ 21.2, 22.9, 24.7, 37.8, 50.3, 55.3,
(54 g, 80% based on starting acid) as an oil: [R]25 -33.8° (c
D
1
0.65, CH3OH); H NMR (CDCl3) δ 0.81 (3H, d, J ) 6.6 Hz),
0.95 (3H, d, J ) 6.3 Hz), 1.57 and 1.75 (2H, m), 1.87 (1H,m),
3.52 (1H, dd, J ) 18, 1.2 Hz), 3.61 (1H, dd, J ) 5.4, 1.2 Hz),
3.63 (3H, s), 3.72 (2H, ABq, J ) 12.9 Hz), 3.81 (3H, s), 3.82
(1H, d, J ) 18 Hz), 5.01 (2H, ABq, J ) 7.0 Hz), 6.86 (2H, d, J
) 9.0 Hz), 7.17 (2H, d, J ) 9.0 Hz); 13C NMR (CDCl3) δ 21.3,
23.0, 24.5, 37.9, 51.2, 55.3, 58.3, 58.3, 62.4, 100.6, 114.0, 128.4,
130.3, 159.4, 167.2, 170.4; HRESI-FTMS calcd for C18H27N2O5
(M + H) 351.1914, found 351.1911. Anal. Calcd for C18H26
-
N2O5: C, 61.70; H, 7.47; N, 7.99. Found: C, 61.85; H, 7.73; N,
8.16.
Syn th esis of 1-Meth oxym eth oxy-3S-ben zyl-4-p-m eth -
oxyben zylp ip er a zin e-2, 6-d ion e (17b). The acid 13b (79.9
mmol), DCC (16.5 g, 79.9 mmol), N-hydroxysuccinimide (9.2
g, 79.9 mmol), and TEA (22.4 mL, 160 mmol) was reacted in
methylene chloride (200 mL) as described above to give
succinimide ester (14b), which was reacted similarly with
hydroxylamine hydrochloride (8.21 g, 120 mmol) for 48 h. The
reaction mixture was concentrated under reduced pressure to
a volume of 100 mL. The precipitated product (18 g) was
collected by filtration and washed with water. The filtrate was
extracted with EtOAc (3 × 400 mL), and the EtOAc layer was
washed with water, dried (Na2SO4), and evaporated under
reduced pressure to give 7 g of additional product 16b (total
25 g, yield 92.0%). Recrystallization from methylene chloride-
hexane gave colorless granules: mp 161-163 °C; [R]25D -49.4°
(c 0.65, MeOH-CH2Cl2); 1H NMR (CDCl3) δ 3.09 (1H, dd, J )
14.1, 9.3 Hz), 3.16 (1H, dd, J ) 14.1, 5.4 Hz), 3.57 (1H, dd, J
) 18, 0.9 Hz), 3.68 (2H, ABq, J ) 13.2 Hz), 3.77 (3H, s), 3.87
(1H, ddd, J ) 9.3, 5.4, 0.9 Hz), 3.97 (1H, d, J ) 18 Hz), 6.73
(2H, d, J ) 8.7 Hz), 6.87 (2H, d, J ) 8.7 Hz), 7.15 (2H, dd, J
) 7.8, 2.4 Hz), 7.28 (3H, m), 8.50 (1H, broad signal, OH); 13C
NMR (CDCl3) δ 35.2, 50.9, 55.3, 58.2, 64.6, 113.9, 127.0, 128.0,
128.5, 129.2, 130.0, 136.7, 159.2, 166.3, 168.5; HRESI-FTMS
calcd for C19H21N2O4 (M + H) 341.1496, found 341.1497. Anal.
Calcd for C19H20N2O4: C, 67.05; H, 5.92; N, 8.23. Found: C,
66.86; H, 5.67; N, 8.20.
A cooled (-40 °C) solution of N-hydroxy compound 16b (23
g, 67.6 mmol) in methylene chloride (200 mL) was reacted with
methoxymethyl chloride (10.3 mL, 135 mmol). The reaction
was worked up and chromatographed in an analogous manner
as described before to give 15 g (57.8%) of the MOM ether 17b
as oil: [R]25D -36.3° (c 1.48, MeOH-CH2Cl2); 1H NMR (CDCl3)
δ 3.09 (1H, dd, J ) 14.4, 9.3 Hz), 3.16 (1H, dd, J ) 14.4, 5.7
Hz), 3.54 (1H, dd, J ) 18.0, 1.2 Hz), 3.64 (3H, s), 3.69 (2H,
ABq, J ) 13.2 Hz), 3.83 (1H, ddd, J ) 9.0, 5.7, 1.2 Hz), 3.92
(1H, d, J ) 18.0 Hz), 5.02 (2H, ABq, J ) 7.2 Hz), 6.75 (2H, d,
J ) 8.7 Hz), 6.90 (2H, d, J ) 8.7 Hz), 7.16 (2H, dd, J ) 7.8,
2.1 Hz), 7.23 (3H, m); 13C NMR (CDCl3) δ 35.2, 51.7, 55.3, 58.0,
58.3, 65.4, 100.9, 113.9, 126.9, 128.1, 128.5, 129.2, 130.0, 136.8,
159.2, 167.1, 169.1; HRESI-FTMS calcd for C21H25N2O5 (M
+ H) 385.1758, found 385.1765. Anal. Calcd for C21H24N2O5:
C, 65.61; H, 6.29; N, 7.29. Found: C, 65.83; H, 6.24; N, 6.99.
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyben zyl-5S-(1R-h yd r oxy-2-m et h ylp r op yl)p ip er a zin e-

Synthesis of Natural Flutimide
J . Org. Chem., Vol. 66, No. 16, 2001 5511
2,6-d ion e (18a ). To a cooled (-78 °C) solution of MOM ether
17a (5.5 g, 15.7 mmol) in THF (60 mL) and HMPA (10 mL)
was added a 1 M THF solution of lithium hexamethyldisilazide
(18.85 mL, 18.85 mmol) under nitrogen over a period of 10
min. The dark yellow solution thus obtained was stirred at
the same temperature for 3 h. Isobutyraldehyde (5.7 mL, 62.8
mmol) was added slowly (10 min) via a syringe. The pale
solution was stirred for 2 h. The reaction was monitored by
TLC (hexanes-EtOAc, 3:1), and after complete consumption
of the starting MOM ether it was quenched with 20 mL of 10%
aqueous NH4Cl. The reaction mixture was allowed to warm
to room temperature and poured onto EtOAc (800 mL). The
organic layer was washed with water (3 × 300 mL), dried
(Na2SO4), and evaporated under reduced pressure to give a
gum that was chromatographed over a silica gel column.
Elution of the column with 5-20% EtOAc in hexane afforded
) 2.7 Hz, OH), 4.02 (1H, d, J ) 7.8 Hz), 5.04 (2H, ABq, J )
7.5 Hz), 5.23 (1H, dd, J ) 7.8, 2.7 Hz), 6.66 (2H, d, J ) 9.0
Hz), 6.70 (2H, d, J ) 9.0 Hz), 7.07 (2H, t, J ) 8.7 Hz), 7.31
(2H, dd, J ) 8.7, 5.4 Hz); 13C NMR (CDCl3) δ 20.7, 23.2, 23.9,
37.1, 52.0, 55.3, 58.4, 59.7, 63.9, 72.2, 100.8, 113.9, 115.4 (d, J
) 25.7 Hz), 127.7, 129.1 (d, J ) 9.8 Hz), 130.2, 135.2 (d, J )
3.8 Hz), 159.3, 162.8 (d, J ) 294.8 Hz), 169.8, 170.1; HRESI-
FTMS calcd for
C25H32FN2O6 (M + H) 475.2238, found
475.2222. Anal. Calcd for C25H31FN2O6: C, 63.28; H, 6.58; N,
5.90. Found: C, 63.52; H, 6.47; N, 5.72.
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyben zyl-5S-[(m -flu or op h en yl)-1R-h yd r oxym eth yl]p ip -
er a zin e-2,6-d ion e (18e). Reaction of MOM ether 17a (1.75
g, 5 mmol) with m-fluorobenzaldehyde (1.06 mL, 10 mmol),
as described above, gave 0.95 g (40%) of R-hydroxy m-fluoro
compound 18e as a gum: [R]25 -20.7° (c 1.61, MeOH); 1H
D
4.2 g (63.3%) of aldol product 18a as a gum: [R]25 -24.2° (c
D
NMR (CDCl3) δ 0.44 (3H, d, J ) 6.6 Hz), 0.82 (3H, d, J ) 6.3
Hz), 1.44 (2H, m), 1.80 (1H, m), 3.23 (1H, d, J ) 12.9 Hz),
3.58 (1H, dd, J ) 10.8, 4.2 Hz), 3.63 (3H, s), 3.73 (3H, s), 3.85
(1H, d, J ) 12.9 Hz), 3.99 (1H, d, J ) 7.8 Hz), 5.02 (2H, ABq,
J ) 7.5 Hz), 5.22 (1H, d, J ) 7.5 Hz), 6.70 (4H, brs), 7.07 (3H,
m), 7.30 (1H, m); HRESI-FTMS calcd for C25H32FN2O6 (M +
H) 475.2238, found 475.2227. Anal. Calcd for C25H31FN2O6: C,
63.28; H, 6.58; N, 5.90. Found: C, 63.18; H, 6.51; N, 5.55.
0.54, MeOH); 1H NMR (C6D6) δ 0.52 (3H, d, J ) 6.5 Hz), 0.74
(3H, d, J ) 7.0 Hz), 0.88 (3H, d, J ) 6.5 Hz), 1.03 (3H, d, J )
7.0 Hz), 1.41 (2H, t, J ) 7.5 Hz), 1.73 (1H,nonet, J ) 6.5 Hz),
2.10 (1H, doublet of heptet, J ) 7.0, 3.5 Hz), 3.19 (1H, d, J )
13 Hz), 3.25 (3H, s), 3.30 (1H, brs, OH), 3.49 (3H, s), 3.58 (1H,
d, J ) 13 Hz), 3.62 (1H, d, J ) 7.5 Hz), 3.72 (1H, t, J ) 7.5
Hz), 3.91 (1H, brdd, J ) 7.0, 2.5 Hz), 4.92 (1H, d, J ) 7.5 Hz),
5.02 (1H, d, J ) 7.5 Hz), 6.66 (2H, d, J ) 8.5 Hz), 6.98 (2H, d,
J ) 9.0 Hz); HRESI-FTMS calcd for C22H35N2O6 423.2489,
found 423.2500. Anal. Calcd for C22H34N2O6: C, 62.54; H, 8.11;
N, 6.62. Found: C, 62.65; H, 8.36; N, 6.33.
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyb en zyl-5S-[(o-flu or op h en yl)-1R- a n d -1S-h yd r oxy-
m eth yl]p ip er a zin e-2,6-d ion e (18f a n d 19f). MOM ether 17a
(1.75 g, 5 mmol) and o-fluorobenzaldehyde (1.05 mL, 10 mmol)
were reacted as described above to give after chromatography
220 mg of S-hydroxy (19f) and 570 mg of R-hydroxy (18f)
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyben zyl-5S-[(p h en yl)-1R-h yd r oxym eth yl]p ip er a zin e-
2,6-d ion e (18b). The aldol reaction performed with MOM
ether 17a (3.5 g, 10 mmol) and benzaldehyde (2.03 mL, 20
mmol), as described above, gave 3.08 g (67.5%) of R-hydroxy
compounds (combined yield 33.3%) both as a gum. 18f: [R]25
D
1
-22.8° (c 0.93, MeOH); H NMR (CDCl3) δ 0.34 (3H, d, J ) 6
Hz), 0.81 (3H, d, J ) 6 Hz), 1.38 (2H, m), 1.76 (1H, m), 3.24
(1H, d, J ) 13.9 Hz), 3.54 (1H, dd, J ) 10.5, 3.6 Hz), 3.65 (3H,
s), 3.74 (3H, s), 3.75 (1H, brd, J ) 3.3 Hz, OH), 3.85 (1H, d, J
) 12.6 Hz), 4.25(1H, d, J ) 8.1 Hz), 5.05 (2H, ABq, J ) 7.5
Hz), 5.59 (1H, dd, J ) 8.1, 3.0 Hz), 6.60 (2H, d, J ) 9.0 Hz),
6.68 (2H, d, J ) 9.0 Hz), 7.10 (1H, m), 7.19 (1H, dt, J ) 7.5,
2.1 Hz), 7.40 (2H, m); 13C NMR (CDCl3) δ 20.6, 23.2, 23.9, 37.3,
52.3, 55.2, 58.4, 60.1, 62., 67.7, 100.8, 113.8, 115.7 (d, J ) 26.5
Hz), 124.4 (d, J ) 4.1 Hz), 126.3 (d, J ) 15.5 Hz), 127.9, 129.3
(d, J ) 4.0 Hz), 130.2 (d, J ) 10.3 Hz), 130.3, 159.2, 160.9 (d,
compound 18b as a foam: [R]25 -17.7° (c 1.9, MeOH); 1H
D
NMR (CDCl3) δ 0.37 (3H, d, J ) 6.0 Hz), 0.81 (3H, d, J ) 6.3
Hz), 1.43 (3H, m), 3.23 (1H, d, J ) 12.6 Hz), 3.52 (1H, dd, J )
10.5, 3.6 Hz), 3.65 (3H, s), 3.73 (3H, s), 3.78 (1H, brs, OH),
3.87 (1H, d, J ) 12.6 Hz), 4.09 (1H, d, J ) 7.8 Hz), 5.05 (2H,
ABq, J ) 7.5 Hz), 5.26 (1H, dd, J ) 7.8, 1.2 Hz), 6.62 (2H, d,
J ) 8.7 Hz), 6.68 (2H, d, J ) 9 Hz), 7.38 (5H, m); 13C NMR
(CDCl3) δ 20.7, 23.3, 23.8, 37.1, 52.0, 55.2, 58.4, 59.8, 63.8,
72.9, 100.8, 113.8, 127.5, 127.9, 128.5, 128.7, 130.3, 139.1,
159.2, 169.9, 170.3; HRESI-FTMS calcd for C25H33N2O6 (M
+ H) 457.2333, found 457.2328. Anal. Calcd for C25H32N2O6:
C, 65.77; H, 7.07; N, 6.14. Found: C, 65.61; H, 6.80; N, 6.01.
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyb en zyl-5S-[(p -m et h oxyp h en yl)-1R-h yd r oxym et h yl]-
p ip er a zin e-2,6-d ion e (18c). Under reaction conditions iden-
tical to those described above, MOM ether 17a (1.55 g, 4.42
mmol) was reacted with p-methoxybenzaldehyde and LHMDS
in THF-HMPA and chromatographed over silica gel column
J ) 294.3 Hz), 169.9, 170.3; HRESI-FTMS calcd for C25H32
-
FN2O6 (M + H) 475.2238, found 475.2228. Anal. Calcd for
C
25H31FN2O6: C, 63.28; H, 6.58; N, 5.90. Found: C, 63.13; H,
6.42; N, 5.80. 19f: [R]25 -49.2° (c 0.61, MeOH); 1H NMR
D
(CDCl3) δ 0.78 (3H, brd, J ) 6 Hz), 0.94 (3H, d, J ) 6 Hz),
1.28 (2H, m), 1.73 (1H, m), 3.21 (1H, d, J ) 13.2 Hz), 3.45
(1H, m), 3.64 (3H, s), 3.78 (3H, s), 4.00 (1H, d, J ) 5.4 Hz),
4.10 (1H, d, J ) 13.2 Hz), 4.17 (1H, brs, OH), 5.03 (2H, ABq,
J ) 7.2 Hz), 5.63 (1H, dd, J ) 5.7, 2.7 Hz), 6.70 (2H, d, J )
8.4 Hz), 6.76 (2H, d, J ) 8.4 Hz), 6.95 (1H, dd, J ) 10.5, 8.1
Hz), 7.20 (1H, dt, J ) 7.2, 0.6 Hz), 7.30 (1H, m), 7.53 (1H, dt,
J ) 7.5, 1.5 Hz); HRESI-FTMS calcd for C25H32FN2O6 (M +
H) 475.2238, found 475.2219. Anal. Calcd for C25H31FN2O6: C,
63.28; H, 6.58; N, 5.90. Found: C, 63.23 H, 5.99; N, 5.63.
to give R-hydroxy product 18c (1.2 g, 56%) as a gum: [R]25
D
1
-1.76° (c 1.02, MeOH); H NMR (CDCl3) δ 0.40 (3H, d, J )
6.0 Hz), 0.83 (3H, d, J ) 6.3 Hz), 1.44 (2H, m), 1.75 (1H, m),
3.23 (1H, d, J ) 13.8 Hz), 3.53 (1H, dd, J ) 10.5, 3.9 Hz), 3.66
(3H, s), 3.75 (3H, s), 3.85 (3H, s), 3.87 (1H, d, J ) 12.9 Hz),
4.08 (1H, d, J ) 7.8 Hz), 5.05 (2H, ABq, J ) 7.5 Hz), 5.24 (1H,
d, J ) 7.8 Hz), 6.66 (2H, d, J ) 9.0 Hz), 6.71 (2H, d, J ) 9.0
Hz), 6.93 (2H, d, J ) 9.0 Hz), 7.29 (2H, d, J ) 8.7 Hz); 13C
NMR (CDCl3) δ 20.7, 23.3, 23.8, 37.2, 52.0, 55.3, 55.5, 58.4,
59.8, 63.8, 72.5, 100.8, 113.8, 113.9, 127.9, 128.7, 130.3, 131.4,
159.2, 159.8, 170.0, 170.2; FABMS m/z 487 (M + H), 493 (M
+ Li); HRESI-FTMS calcd for C26H35N2O7 (M + H) 487.2438,
found 487.2414. Anal. Calcd for C26H34N2O7: C, 64.18; H, 7.04;
N, 5.76. Found: C, 64.52; H, 6.89; N, 5.52.
Syn th esis of 1-Meth oxym eth oxy-3S-ben zyl-4-p-m eth -
oxyb en zyl-5S-[(p -m et h oxyp h en yl)-1R-h yd r oxym et h yl]-
p ip er a zin e-2,6-d ion e (18h ). Reaction of the MOM ether 17b
(2.5 g, 6.5 mmol) with p-methoxybenzaldehyde (2 equiv) after
chromatography gave R-hydroxy product 18h (1.2 g, 56%) as
1
a gum: [R]25 -22.4° (c 1.2, MeOH); H NMR (CDCl3) δ 2.94
D
(1H, dd, J ) 14.7, 10.5 Hz), 3.16 (1H, dd, J ) 14.4, 4.2 Hz),
3.27 (1H, d, J ) 12.9 Hz), 3.67 (3H, s), 3.74 (3H, s), 3.76 (1H,
d, J ) 12.9 Hz), 3.84 (3H, s), 3.86 (1H, dd, J ) 10.8, 4.5 Hz),
3.97 (d, J ) 13.2 Hz), 4.01 (1H, d, J ) 7.5 Hz), 5.08 (2H, ABq,
J ) 7.5 Hz), 5.14 (1H, brd, J ) 7.5 Hz), 6.43 (2H, d, J ) 8.7
Hz), 6.56 (2H, d, J ) 9.0 Hz), 6.79 (2H, d, J ) 8.7 Hz), 6.8
(2H, d, J ) 8.1 Hz), 7.07-7.22 (5H, m); 13C NMR (CDCl3) δ
34.1, 52.0, 55.2, 55.4, 58.5, 63.1, 64.9, 71.9, 100.9, 113.7, 113.9,
126.6, 127.3, 128.3, 128.3, 129.3, 130.0, 131.4, 136.5, 159.0,
159.6, 169.0, 169.8; HRESI-FTMS calcd for C29H33N2O7 (M
+ H) 521.2282, found 521.2287. Anal. Calcd for C29H32N2O7:
C, 66.91; H, 6.20; N, 5.38. Found: C, 66.81; H, 6.03; N, 5.30.
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyben zyl-5S-[(p-flu or op h en yl)-1R-h yd r oxym eth yl]p ip -
er a zin e-2,6-d ion e (18d ). The aldol reaction was repeated
with MOM ether 17a (3.5 g, 10 mmol) and p-fluorobenzalde-
hyde (2.15 mL, 20 mmol), as described above, to give 3.17 g
(67.5%) of R-hydroxy fluoro compound 18d as a foam: [R]25
D
1
-18.8° (c 2.87, MeOH); H NMR (CDCl3) δ 0.41 (3H, d, J )
6.0 Hz), 0.82 (3H, d, J ) 6.6 Hz), 1.43 (2H, m), 1.71 (1H, m),
3.23 (1H, d, J ) 12.9 Hz), 3.54 (1H, dd, J ) 10.5, 3.9 Hz), 3.64
(3H, s), 3.74 (3H, s), 3.83 (1H, d, J ) 12.6 Hz), 3.84 (1H, d, J

5512 J . Org. Chem., Vol. 66, No. 16, 2001
Singh and Tomassini
Syn th esis of 1-Meth oxym eth oxy-3S-ben zyl-4-p-m eth -
oxyben zyl-5S-[(p -flu or op h en yl)-1R-h yd r oxym eth yl]p ip -
er a zin e-2,6-d ion e (18i). Reaction of the MOM ether 17b (1.9
g, 4.95 mmol) with p-fluorobenzaldehyde (1.5 equiv) after
chromatography gave R-hydroxy product 18i (1.62 g, 64.5%)
38.3 (CH2), 55.3 (OCH3), 56.3 (CH2), 58.2 (OCH3), 60.2 (CH),
100.6 (OCH2O), 113.9 (2 × CH), 128.2, 130.2 (2 × CH), 130.4,
143.6 (CH), 159.2, 160.3, 169.3; FABMS m/z 427 (M + Na),
405 (M + H).
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyben zyl-5-Z-ben zylid en ep ip er a zin e-2,6-d ion e (21b). R-
Hydroxy benzyl compound 18b (1.42 g, 3.1 mmol) was reacted
with methanesulfonyl chloride (2 equiv) under identical condi-
tions as described above to give mesylate 20b, which sponta-
as a foam that turned into a gum: [R]25 -27.7° (c 1.24,
D
1
MeOH); H NMR (CDCl3) δ 2.94 (1H, dd, J ) 14.7, 10.8 Hz),
3.20 (1H, dd, J ) 15.0, 4.5 Hz), 3.28 (1H, d, J ) 13.2 Hz), 3.67
(3H, s), 3.74 (3H, s), 3.89 (1H, dd, J ) 10.8, 4.5 Hz), 3.956
(1H, d, J ) 7.8 Hz), 3.957 (1H, d, J ) 13.2 Hz), 5.08 (2H, ABq,
J ) 7.2 Hz), 5.13 (1H, dd, J ) 7.8, 1.8 Hz), 6.41 (2H, d, J )
8.4 Hz), 6.57 (2H, d, J ) 8.7 Hz), 6.84 (2H, dd, J ) 6.9, 1.5
Hz), 6.91 (2H, dd, J ) 8.4, 5.4 Hz), 7.23 (3H, m); 13C NMR
(CDCl3) δ 34.0, 52.0, 55.2, 58.5, 63.1, 64.9, 71.6, 100.9, 113.7,
115.2 (d, J ) 25.7 Hz), 126.8, 127.1, 128.4, 128.9 (d, J ) 9.8
Hz), 129.2, 129.9, 135.1 (d, J ) 3.9 Hz), 136.5, 159.1, 162.6 (d,
neously produced Z-olefin 21b (1.0 g, 73.3%) as a yellow gum:
1
[R]25 +27.5° (c 0.16, MeOH); H NMR (CDCl3) δ 0.83 (3H, d,
D
J ) 6.6 Hz), 0.87 (3H, d, J ) 6.6 Hz), 1.47 (1H, ddd, J ) 14.1,
8.7, 5.1 Hz), 1.64 (1H, ddd, J ) 13.8, 9.9, 5.4 Hz), 1.84 (1H,
m), 3.60 (3H, s), 3.72 (1H, dd, J ) 9.9, 5.1 Hz), 3.78 (3H, s),
3.95 (2H, ABq, J ) 13.8 Hz), 4.80 (2H, ABq, J ) 7.2 Hz), 6.83
(2H, d, J ) 8.7 Hz), 7.12 (2H, d, J ) 8.7 Hz), 7.44 (3H, m),
7.56 (1H, s), 7.96 (2H, dd, J ) 8.7, 1.8 Hz); 13C NMR (CDCl3)
δ 21.4 (CH3), 23.0 (CH3), 24.8 (CH), 42.7 (CH2), 55.3 (OCH3),
58.1 (CH), 59.0 (CH2), 60.1 (OCH3), 100.9 (OCH2O), 114.2 (2
× CH), 127.7, 128.8 (2 × CH), 129.9 (CH), 130.5 (2 × CH),
130.9 (2 × CH), 131.1 (CH), 133.3, 133.8, 159.6, 161.3, 170.0;
HRESI-FTMS calcd for C25H31N2O5 (M + H) 439.2227, found
439.2224.
J ) 294.4 Hz), 168.8, 169.6; HRESI-FTMS calcd for C28H30
FN2O6 (M + H) 509.2082, found 509.2080. Anal. Calcd for
28H29N2O6: C, 66.13; H, 5.75; N, 5.51. Found: C, 66.43; H,
-
C
5.74; N, 5.53.
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyben zyl-5-(2-m eth yl-Z- a n d -E-p r op -1-en yl)p ip er a zin e-
2,6-d ion e (21a a n d 22a ). To a cooled (-23 °C) solution of
hydroxy compound 18a (3.8 g, 9 mmol) in methylene chloride
(50 mL) were added DIPEA (3.13 mL, 18 mmol) and DMAP
(2.2 g, 18 mmol) under nitrogen. After 10 min, methanesulfonyl
chloride (1.39 mL, 18 mmol) was slowly added via a syringe,
and the mixture was stirred for 20 min at -23 °C followed by
1 h at room temperature. The reaction mixture was conve-
niently monitored on TLC (hexanes-EtOAc 7:3) and formation
of a slightly polar product was observed. Water (100 mL) was
added to the mixture and extracted with EtOAc (800 mL). The
EtOAc layer was washed sequentially with water (2 × 200
mL), 10% aqueous citric acid (2 × 200 mL), water (200 mL),
20% aqueous NaHCO3 (2 × 200 mL), and finally with water
(2 × 200 mL), dried over Na2SO4, and evaporated to give
chromatographically homogeneous mesylate 20a (4.5 g) as a
gum that was used without purification.
To a cooled (0 °C) solution of the mesylate (4.5 g, 9 mmol)
in toluene (25 mL) was added DBU (4.0 mL, 27 mmol) via a
syringe under nitrogen. The solution was stirred at 0 °C for
10 min, room temperature for 30 min, and at 50 °C for 1 h.
The reaction mixture was allowed to cool to room temperature
before adding EtOAc (700 mL). The EtOAc solution was
sequentially washed with water, aqueous citrtic acid, water,
aquoues NaHCO3, and water, dried over Na2SO4, and evapo-
rated under reduced pressure to give the crude product as a
gum that was chromatographed over a silica gel column and
eluted with 2-15% of EtOAc in hexane to give 400 mg of
E-isomer 22a , 800 mg of a mixture of E and Z isomers, and
finally 1.53 g of Z-isomer 21a all as gums. The ismeric ratio
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyben zyl-5-Z-p-m eth oxyben zylid en ep ip er a zin e-2,6-d i-
on e (21c). To a cooled (-23 °C) and stirred solution of the
hydroxy compound 18c (720 mg, 1.48 mmol) in methylene
chloride (10 mL) were added DIPEA (0.52 mL, 2.96 mmol) and
DMAP (361 mg, 2.96 mmol) followed by slow addition of
methanesulfonyl chloride (0.23 mL, 2.96 mmol) via a syringe.
The reaction mixture was stirred for 30 min at -23 °C followed
by stirring at room temperature for 1 h. The mesylate (20c)
formed in this reaction was short-lived and produced almost
instantaneously the elimination product (21c). The mixture
was poured on to EtOAc (200 mL) and was washed with water,
10% aqueous citric acid, water, aqueous NaHCO3, and finally
with water. The organic layer was dried over Na2SO4, evapo-
rated under reduced pressure, and chromatographed over a
silica gel column. Elution with 5-20% EtOAc in hexane
afforded (528 mg, 76%) pure product 21c as a yellow gum:
1
[R]25 0° (c 0.98, MeOH); H NMR (CDCl3) δ 0.80 (3H, d, J )
D
6.6 Hz), 0.84 (3H, d, J ) 6.6 Hz), 1.43 (1H, ddd, J ) 13.8, 9.0,
5.1 Hz), 1.62 (1H, ddd, J ) 13.8, 10.2, 5.4 Hz), 1.81 (1H, m),
3.59 (3H, s), 3.70 (1H, dd, J ) 10.2, 5.4 Hz), 3.78 (3H, s), 3.88
(3H, s), 3.93 (2H, ABq, J ) 13.8 Hz), 4.80 (2H, ABq, J ) 7.2
Hz), 6.84 (2H, d, J ) 8.7 Hz), 6.98 (2H, d, J ) 8.7 Hz), 7.15
(2H, d, J ) 8.4 Hz), 7.54 (1H, s), 7.99 (2H, d, J ) 9.0 Hz); 13
C
NMR (CDCl3) δ 21.3, 23.0, 24.8, 42.3, 55.3, 55.4, 58.0, 58.7,
60.0, 100.8, 114.2, 114.2, 126.5, 127.8, 130.7, 131.0, 132.1,
133.0, 159.6, 161.0, 161.6, 170.2; FABMS m/z 475 (M + Li);
HRESI-FTMS calcd for C26H33N2O6 (M + H) 479.2333, found
469.2319. Anal. Calcd for C26H32N2O6: C, 66.65; H, 6.88; N,
5.98. Found: C, 66.65; H, 6.80; N, 5.92.
Z/E was measured to be ∼3/1 and overall two step yield 74.3%.
1
21a : [R]25 +37.6° (c 0.21, CH3OH); H NMR (CDCl3) δ 0.63
D
(3H, d, J ) 6.4 Hz), 0.84 (3H, d, J ) 6.8 Hz), 0.99 (3H, d, J )
6.8 Hz), 1.03 (3H, d, J ) 6.8 Hz), 1.29 (1H, ddd, J ) 14, 9.6,
4.4 Hz), 1.49 (1H, ddd, J ) 13.6, 10.8, 4.4 Hz), 1.77 (1H,m),
3.11 (1H, m), 3.55 (1H, dd, J ) 11.2, 4.4 Hz), 3.62 (3H, s), 3.78
(1H, d, J ) 12.8 Hz), 3.79 (3H, s), 3.81 (2H, s), 4.95 (2H, ABq,
J ) 7.2 Hz), 6.71 (1H, d, J ) 11.2 Hz), 6.86 (2H, d, J ) 8.4
Hz), 7.20 (2H, d, J ) 8.8 Hz); 13C NMR (CDCl3) δ 20.8 (CH3),
21.3 (CH3), 22.1 (CH3), 23.1 (CH3), 24.1 (CH), 26.8 (CH), 41.1
(CH2), 55.3 (OCH3), 58.1 (CH2), 60.0 (OCH3), 60.8 (CH), 100.7
(OCH2O), 114.0 (2 × CH), 128.3, 130.6 (2 × CH), 132.7, 147.6
(CH), 159.4, 160.8, 170.78: FABMS m/z 427 (M + Na), 405
(M + H). Anal. Calcd for C22H32N2O5: C, 65.31; H, 7.97; N,
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyb e n zyl-5-Z-p -flu or ob e n zylid e n e p ip e r a zin e -2,6-d i-
on e (21d ). An elimination reaction similar to the one just
described from R-hydroxy p-fluorobenzyl compound 18d (1.42
g, 3.1 mmol) via mesylate 20d gave Z-olefin 21d (1.05 g, 78%)
as a yellow gum: [R]25 +12.3° (c 1.32, MeOH); 1H NMR
D
(CDCl3) δ 0.82 (3H, d, J ) 6.6 Hz), 0.85 (3H, d, J ) 6.6 Hz),
1.45 (1H, ddd, J ) 14.1, 8.7, 5.4 Hz), 1.61 (1H, ddd, J ) 13.8,
9.6, 5.4 Hz), 1.77 (1H, m), 3.58 (3H, s), 3.71 (1H, dd, J ) 9.6,
5.4 Hz), 3.77 (3H, s), 3.92 (2H, ABq, J ) 13.8 Hz), 4.80 (2H,
ABq, J ) 7.2 Hz), 6.82 (2H, d, J ) 8.7 Hz), 7.11 (2H, d, J )
8.7 Hz), 7.14 (2H, t, J ) 8.4 Hz), 7.51 (1H, s), 7.98 (2H, dd, J
) 8.7, 5.4 Hz); 13C NMR (CDCl3) δ 21.4, 22.9, 24.8, 42.5, 55.3,
58.1, 58.9, 60.1, 100.8, 114.2, 115.9 (d, J ) 25.8 Hz), 127.5,
130.0 (d, J ) 4.3 Hz), 130.2, 130.6 (2C), 133.0 (d, J ) 9.8 Hz),
159.6, 161.3, 163.2 (d, J ) 301.2 Hz), 169.9; HRESI-FTMS
calcd for C25H30FN2O5 (M + H) 457.2133, found 457.2109. Anal.
Calcd for C25H29FN2O5‚0.5H2O: C, 64.51; H, 6.49; N, 6.01.
Found: C, 64.29; H, 6.16; N, 5.95.
6.92. Found: C, 65.61; H, 7.65; N, 6.63. E-isomer 22a : [R]25
D
+76° (c 0.25, CH3OH); 1H NMR (CDCl3) δ 0.81 (3H, d, J ) 6.4
Hz), 0.83 (3H, d, J ) 6.4 Hz), 0.93 (3H, d, J ) 6.4 Hz), 0.98
(3H, d, J ) 6.4 Hz), 1.41 (1H, ddd, J ) 14, 7.0, 7.0 Hz), 1.50
(1H, ddd, J ) 14, 7.0, 7.0 Hz), 1.74 (1H, nonet, J ) 6.4 Hz),
3.58 (1H, m), 3.63 (3H, s), 3.65 (1H, t, J ) 8.0 Hz), 3.78 (1H,
d, J ) 12.8 Hz), 3.79 (3H, s), 4.00 (1H, d, J ) 13.2 Hz), 5.00
(2H, ABq, J ) 7.2 Hz), 5.44 (1H, d, J ) 10 Hz), 6.84 (2H, d, J
) 8.8 Hz), 7.15 (2H, d, J ) 8.4 Hz); 13C NMR (CDCl3) δ 22.2
(CH3), 22.4 (CH3), 22.7 (CH3), 23.1 (CH3), 24.3 (CH), 26.6 (CH),
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyb en zyl-5-Z-m -flu or ob en zylid en ep ip er a zin e-2,6-d i-
on e (21e). Elimination reaction of hydroxy-m-fluorobenzyl

Synthesis of Natural Flutimide
J . Org. Chem., Vol. 66, No. 16, 2001 5513
compound 18e (0.75 g, 1.58 mmol) gave Z-olefin 21e (0.53 g,
73.5%) as a yellow gum: [R]25D +44.8° (c 0.67, MeOH); 1H NMR
(CDCl3) δ 0.84 (3H, d, J ) 6.6 Hz), 0.88 (3H, d, J ) 6.6 Hz),
1.47 (1H, ddd, J ) 14.1, 8.7, 5.4 Hz), 1.62 (1H, ddd, J ) 14.1,
9.9, 5.4 Hz), 1.81 (1H, m), 3.60 (3H, s), 3.73 (1H, dd, J ) 9.9,
5.4 Hz), 3.77 (3H, s), 3.90 (1H, d, J ) 13.5 Hz), 3.98 (1H, d, J
) 13.5 Hz), 4.80 (2H, ABq, J ) 7.2 Hz), 6.83 (2H, d, J ) 8.7
Hz), 7.10 (1H, m), 7.11 (2H, d, J ) 8.7 Hz), 7.41 (1H, dt, J )
8.1, 6.0 Hz), 7.49 (1H, s), 7.57 (1H, d, J ) 7.5 Hz), 7.83 (1H,
ddd, J ) 10.5, 2.4, 1.5 Hz); 13C NMR (CDCl3) δ 21.4, 22.9, 24.9,
42.8, 55.3, 58.1, 59.2, 60.1, 100.9, 114.2, 116.7 (d, J ) 27.4
Hz), 116.7 (d, J ) 27.4 Hz), 127.1 (d, J ) 3.3 Hz), 127.4, 129.24
(d, J ) 3.4 Hz), 130.2 (d, J ) 10 Hz), 130.3, 134.5, 135.8 (d, J
) 9.9 Hz), 159.7, 161.0, 162.8 (d, J ) 293.2 Hz), 169.7; HRESI-
s), 3.83 (3H, s), 3.87 (1H, d, J ) 14.1 Hz), 4.00 (1H, dd, J )
10.8, 3.9 Hz), 4.83 (2H, ABq, J ) 7.2 Hz), 6.77 (2H, d, J ) 8.7
Hz), 6.78 (2H, d, J ) 9.0 Hz), 6.93 (2H, d, J ) 8.7 Hz), 7.03
(1H, dd, J ) 7.5, 1.2 Hz), 7.18-7.24 (5H, m), 7.54 (1H, s), 7.59
(2H, d, J ) 8.7 Hz); 13C NMR (CDCl3) δ 39.5, 55.3, 55.4, 58.1,
58.6, 63.5, 100.9, 113.9, 114.1, 126.1, 126.9, 127.7, 128.5, 129.5,
130.2, 130.3, 131.7, 133.4, 136.7, 159.5, 160.9, 161.5, 169.3;
HRESI-FTMS calcd for C29H31N2O6 (M + H) 503.2176, found
503.2158. Anal. Calcd for C29H30N2O6‚0.8 H2O: C, 67.38; H,
6.16; N, 5.41. Found: C, 67.42; H, 5.84; N, 5.60.
Syn th esis of 1-Meth oxym eth oxy-3S-ben zyl-4-p-m eth -
oxyb e n zyl-5-Z-p -flu or ob e n zylid e n e p ip e r a zin e -2,6-d i-
on e (21i). In an identical reaction condition starting with
hydroxy compound 18i (1.2 g, 2.36 mmol), the elimination
FTMS calcd for
C
25H30FN2O5 (M + H) 457.2133, found
reaction gave product 21i as a yellow powder (0.81 g, 70.7%):
1
457.2143. Anal. Calcd for C25H29FN2O5‚0.5 H2O: C, 64.51; H,
6.49; N, 6.01. Found: C, 64.91; H, 6.26; N, 6.08.
[R]25 + 10.7° (c 0.48, MeOH-CH2Cl2, 1:1); H NMR (CDCl3)
D
δ 2.83 (1H, dd, J ) 14.7, 10.8 Hz), 3.17 (1H, dd, J ) 14.1, 4.2
Hz), 3.61 (3H, s), 3.70 (1H, d, J ) 14.1 Hz), 3.86 (1H, d, J )
14.4 Hz), 4.05 (1H, dd, J ) 11.4, 4.2 Hz), 4.84 (2H, ABq, J )
6.9 Hz), 6.78 (2H, d, J ) 8.7 Hz), 6.92 (2H, d, J ) 8.7 Hz),
6.93 (2H, t, J ) 9 Hz), 7.05 (1H, dd, J ) 8.1, 1.2 Hz), 7.20-
7.33 (3H, m), 7.49 (1H, s), 7.55 (2H, dd, J ) 9.9, 5.7 Hz), 7.63
(1H, dd, J ) 7.5, 1.5 Hz); HRESI-FTMS calcd for C28H27FN2O5
Syn th esis of 1-Meth oxym eth oxy-3S-isobu tyl-4-p-m eth -
oxyb e n zyl-5-Z-o-flu or ob e n zylid e n e p ip e r a zin e -2,6-d i-
on e (21f). Hydroxy-o-fluorobenzyl compound 18f (0.42 g, 0.9
mmol) gave Z-olefin 21f (0.4 g, 99%) as a yellow gum: [R]25
D
+11.5° (c 0.6, MeOH); 1H NMR (CDCl3) δ 0.84 (3H, d, J ) 6.6
Hz), 0.89 (3H, d, J ) 6.6 Hz), 1.49 (1H, ddd, J ) 13.8, 8.4, 5.4
Hz), 1.65 (1H, ddd, J ) 13.8, 9.6, 5.7 Hz), 1.81 (1H, m), 3.59
(3H, s), 3.72 (1H, dd, J ) 9.6, 5.7 Hz), 3.77 (3H, s), 3.87 (1H,
d, J ) 13.8 Hz), 3.99 (1H, d, J ) 13.8 Hz), 4.83 (2H, ABq, J )
7.2 Hz), 6.81 (2H, d, J ) 8.7 Hz), 7.07 (2H, d, J ) 8.7 Hz),
7.13 (1H, ddd, J ) 10.2, 8.4, 1.2 Hz), 7.23 (1H, dt, J ) 7.2, 0.9
Hz), 7.36 (1H, m), 7.71 (1H, s), 8.27 (1H, dt, J ) 7.8, 1.8 Hz);
13C NMR (CDCl3) δ 21.6, 22.8, 24.8, 43.0, 55.3, 58.1, 59.0, 60.1,
100.8, 114.2, 115.9 (d, J ) 26.4 Hz), 120.4 (d, J ) 7.4 Hz),
122.2 (d, J ) 13.8 Hz), 124.1 (d, J ) 4.4 Hz), 127.7, 130.1 (d,
J ) 2.3 Hz), 130.3, 131.2 (d, J ) 10.4 Hz), 134.8 (d, J ) 1.9
Hz), 159.6, 160.7, 161.2 (d, J ) 302.5 Hz), 169.6; HRESI-
(M + H) 491.1977, found 491.1963. Anal: Calcd for C28H27
-
FN2O5‚H2O: C, 66.13; H, 5.74; N, 5.50. Found: C, 65.82; H,
5.64; N, 5.65.
Syn th esis of 1-Meth oxym eth oxy-3-isobu tyl-5-(2-m eth -
yl-Z-p r op -1-en yl)-3H-p yr a zin e-2,6-d ion e (23a ). To a solu-
tion of N-p-methoxybenzyl compound 21a (96 mg, 0.24 mmol)
in methylene chloride (5 mL) were added water (2.5 mL) and
DDQ (197 mg, 0.84 mmol, 3.5×), and the reaction mixture was
stirred at room temperature overnight. The reaction mixture
was filtered through a small bed of silica gel and Celite and
eluted with 50% EtOAc-hexane. The product mixture was
rechromatographed over a small silica gel column and eluted
with 5% EtOAc-hexane to give 20 mg (30%) chromatographi-
cally homogeneous imine product 23a as an oil: 1H NMR
(CDCl3) δ 0.99 (3H, d, J ) 6.8 Hz), 1.13 (3H, d, J ) 6.4 Hz),
2.19 (1H, heptet, J ) 6.4 Hz), 2.64 (1H, d, J ) 6.8 Hz), 3.54
(1H, m), 3.66 (3H, s), 5.08 (2H, s), 7.13 (1H, d, J ) 10.4 Hz);
13C NMR (CDCl3) δ 21.9, 22.6, 26.4, 27.4, 41.8, 58.4, 100.7,
133.9, 155.3, 157.5, 158.4, 159.8; FABMS m/z 283 (M + H).
Anal. Calcd for C14H22N2O4: C, 59.56; H, 7.85; N, 9.92.
Found: C, 59.62; H, 7.48; N, 9.60.
FTMS calcd for
457.2124. Anal. Calcd for C25H29FN2O5‚0.25H2O: C, 65.78; H,
6.40; N, 6.14. Found: C, 65.41; H, 6.07; N, 6.02.
C25H30FN2O5 (M + H) 457.2133, found
Syn th esis of 1-Meth oxym eth oxy-3S-ben zyl-4-p-m eth -
oxyben zyl-5-(2-m eth yl-Z- a n d E-p r op -1-en yl)p ip er a zin e-
2,6-d ion e (21g a n d 22b). Aldol condensation of MOM ether
17b (2.7 g, 7 mmol) using 3 equiv of isobutyrlaldehyde after
chromatography gave an inseparable ∼2:1 mixture of R- and
S-hydroxy compounds 18g and 19g (1.8 g, 56%) as a gum that
was used for the elimination reaction without any further
characterization: HRESI-FTMS calcd for C25H33N2O6 (M +
H) 457.2333, found 457.2318. Anal. Calcd for C25H32N2O6: C,
65.77; H, 7.07; N, 6.14. Found: C, 65.44; H, 6.82; N, 5.93.
Mesylation of the mixture of the hydroxy compounds 18g
and 19g (1.6 g, 3.5 mmol) gave corresponding mesylates that
upon elimination reaction using DBU gave a 4:1 mixture of Z
and E isomers, which were separated by chromatography on
silica gel to give Z-isomer 21g (810 mg, total two step yield of
57%) as a gum. E-isomer (22b) was not stable and was not
Sca le-u p Rea ction . N-p-Methoxybenzyl compound 21a (1.5
g, 3.7 mmol) in methylene chloride (40 mL), water (20 mL),
and DDQ (2.1 g, 2.5 equiv) was reacted under similar condi-
tions to give 200 mg (20%) of the product 23a . In this reaction,
the isobutylaldehyde was also present in significant quantities
(NMR).
Syn th esis of 1-Meth oxym eth oxy-3-isobu tyl-5-Z-ben z-
ylid en e-3H-p yr a zin e-2,6-d ion e (23b). To a solution of the
PMB derivative 21b (800 mg, 1.83 mmol) in methylene
chloride (10 mL) and water (2 mL) was added DDQ (1.66 g, 4
eq) and the mixture stirred overnight. The reaction mixture
was filtered, diluted with 300 mL of methylene chloride,
washed with water, 10% aqueous NaHCO3, and water, dried
(Na2SO4), evaporated under reduced pressure, and chromato-
graphed over a silica gel column. Elution with 10% EtOAc in
hexane yielded 128 mg (22.2%) of product 23b as a yellow
powder. 1H NMR spectrum of crude reaction mixture indicated
the presence of a significant amount of benzaldehyde, which
was confirmed by co-TLC. 23b: mp 90-92 °C; 1H NMR
(CDCl3) δ 1.04 (6H, d, J ) 6.6 Hz), 2.28 (1H, nonet, J ) 6.6
Hz), 2.75 (2H, d, J ) 6.9 Hz), 3.69 (3H, s), 5.13 (2H, s), 7.47
(3H, m), 7.83 (1H, s), 8.20 (2H, m); 13C NMR (CDCl3) δ 22.6,
26.5, 42.1, 58.5, 100.7, 128.8, 132.0, 133.0, 133.7, 134.1, 142.8,
155.0, 158.3, 160.5; HRESI-FTMS calcd for C17H21N2O4 (M
+ H) 317.1496, found 317.1495. Anal. Calcd for C17H20N2O4:
C, 64.54; H, 6.37; N, 8.85. Found: C, 64.57; H, 6.32; N, 8.53.
Syn th esis of 1-Meth oxym eth oxy-3-isobu tyl-5-Z-p-m eth -
oxyben zylid en e- 3H-p yr a zin e-2,6-d ion e (23c). PMB de-
rivative 21c (235 mg) was reacted in methylene chloride (10
mL) and water (5 mL) with 3 equiv of DDQ under identical
conditions overnight. The reaction mixture was filtered, diluted
characterized. 21g: [R]25 -0.2° (c 1.55, MeOH); 1H NMR
D
(CDCl3) δ 0.99 (3H, d, J ) 6.9 Hz), 1.09 (3H, d, J ) 6.6 Hz),
2.74 (1H, dd, J ) 13.8, 10.8 Hz), 2.88 (1H, m), 2.98 (1H, dd, J
) 13.8, 4.5 Hz), 3.63 (3H, s), 3.78 (3H, s), 3.78 (1H, dd, J )
masked due to overlap), 3.80 (2H, ABq, J ) 13.8 Hz), 4.96 (2H,
ABq, J ) 7.5 Hz), 6.72 (2H, d, J ) 8.7 Hz), 6.73 (1H, d, J )
10.8 Hz), 6.88 (2H, d, J ) 8.4 Hz), 7.01 (2H, m), 7.25 (3H, m);
13C NMR (CDCl3) δ 21.5, 22.2, 26.7, 38.8, 55.3, 58.2, 61.0, 63.2,
100.8, 114.0, 126.8, 128.0, 128.2, 129.2, 129.9, 132.7, 146.5,
159.3, 160.8, 169.6; HRESI-FTMS calcd for C25H31N2O5 (M
+ H) 439.2227, found 439.2233. Anal. Calcd for C25H30N2O5‚
0.25 H2O: C, 67.78; H, 6.88; N, 6.32. Found: C, 67.53; H, 6.77;
N, 6.46.
Syn th esis of 1-Meth oxym eth oxy-3S-ben zyl-4-p-m eth -
oxyben zyl-5-Z-p-m eth oxyben zylid en ep ip er a zin e-2,6-d i-
on e (21h ). This reaction was performed identical to other
aromatic analogues, and the hydroxy compound 18h (2.0 g,
3.85 mmol) was reacted with methanesulfonyl chloride to give
directly after silica gel chromatography the Z-olefin 21h (1.32
g, 68.4%) as a yellow gum: [R]25D + 77.3° (c 1, MeOH); 1H NMR
(CDCl3) δ 2.83 (1H, dd, J ) 14.1, 11.4 Hz), 3.12 (1H, dd, J )
13.8, 3.9 Hz), 3.61 (3H, s), 3.72 (1H, d, J ) 13.8 Hz), 3.77 (3H,

5514 J . Org. Chem., Vol. 66, No. 16, 2001
Singh and Tomassini
with 200 mL of methylene chloride, washed with water, 10%
aqueous NaHCO3, and water, dried (Na2SO4), evaporated
under reduced pressure, and chromatographed over a silica
gel column. Elution with 10% EtOAc in hexane yielded 45 mg
(26%) of required product 23c as a yellow solid and 5 mg of
product 24 as a gum. 23c: 1H NMR (CDCl3) δ 1.04 (6H, d, J
) 6.9 Hz), 2.26 (1H, nonet, J ) 6.9 Hz), 2.73 (2H, d, J ) 6.9
Hz), 3.68 (3H, s), 3.88 (3H, s), 5.12 (2H, s), 6.96 (2H, d, J )
9.0 Hz), 7.78 (1H, s), 8.20 (2H, d, J ) 8.4 Hz); FABMS m/z
353 (M + Li); HRESI-FTMS calcd for C18H22N2O5 (M + H)
347.1601, found 347.1603. 24: 1H NMR (CDCl3) δ 0.95 (6H,
d, J ) 6.9 Hz), 2.18 (1H, nonet, J ) 6.6 Hz), 2.82 (2H, d, J )
6.6 Hz), 3.80 (3H, s), 4.40 (2H, d, J ) 5.7 Hz), 6.87 (2H, d, J
) 8.7 Hz), 7.21 (2H, d, J ) 8.4 Hz); FABMS m/z 250 [M + H].
Syn th esis of 1-Meth oxym eth oxy-3-isobu tyl-5-Z-p-
flu or oben zylid en e-3H-p yr a zin e-2,6-d ion e (23d ). Oxidation
of PMB derivative 21d (700 mg, 1.5 mmol) in methylene
chloride (10 mL) and water (2 mL) with DDQ (1.60 g, 4 equiv)
overnight followed by filtration through silica gel and Celite
and washing of the methylene chloride solution with aqueous
sodium bicarbonate and crystallization of the residue from
acetone-hexane afforded (180 mg, 35.1%) desired product 23d
for C17H20N2O4‚0.5 H2O: C, 62.76; H, 6.50; N, 8.61. Found:
C, 62.67; H, 6.10; N, 8.43. 25: 1H NMR (CDCl3) δ 3.79 (3H, s),
4.24 (2H, s), 4.39 (2H, d, J ) 6.0 Hz), 6.86 (2H, d, J ) 8.7 Hz),
7.18 (2H, d, J ) 8.7 Hz), 7.24-7.34 (5H, m); 13C NMR (CDCl3)
δ 43.1, 43.2, 55.3, 114.3, 127.3, 128.7, 128.9, 129.4, 129.9, 132.7,
159.3, 159.8, 196.0.
Syn th esis of 1-Meth oxym eth oxy-3-ben zyl-5-Z-p-m eth -
oxyben zylid en e-3H-p yr a zin e-2,6-d ion e (23h ). DDQ oxida-
tion of 1.1 g of the PMB derivative (21h ) under the conditions
described above and crystallization from acetone-hexane gave
(400 mg, 48%) of desired product 23h as a yellow granular
1
solid: mp 160-162 °C; H NMR (CDCl3) δ 3.70 (3H, s), 3.86
(3H, s), 4.18 (2H, s), 5.14 (2H, s), 6.79 (2H, d, J ) 9.0 Hz),
7.34 (5H, m), 7.72 (1H, s), 7.86 (2H, d, J ) 9.0 Hz); 13C NMR
(CDCl3) δ 39.8, 55.5, 58.5, 100.7, 114.4, 126.7, 126.8, 128.6,
130.1, 131.0, 136.0, 136.8, 143.5, 155.0, 155.8, 160.5; HRESI-
FTMS calcd for C21H21N2O5 (M + H) 381.1445, found 381.1446.
Anal. Calcd for C21H20N2O5‚0.5H2O: C, 64.77; H, 5.43; N, 7.19.
Found: C, 65.02; H, 5.18; N, 7.20.
Syn th esis
of
1-Meth oxym eth oxy-3-ben zyl-5-Z-p-
flu or oben zylid en e-3H-p yr a zin e-2,6-d ion e (23i). DDQ oxi-
dation of protected compound 21i (0.70 g, 1.43 mmol) followed
by trituration by acetone-hexane gave imine product 23i (150
mg, 28.5%) as a yellow powder: mp 142-144 °C; 1H NMR
(CDCl3) δ 3.69 (3H, s), 4.20 (2H, s), 5.13 (2H, s), 6.94 (2H, t, J
) 9.0 Hz), 7.27-7.43 (5H, m), 7.70 (1H, s), 7.85 (2H, dd, J )
9,0, 5.7 Hz); HRESI-FTMS calcd for C20H18FN2O4 (M + H)
369.1245, found 369.1242. Anal. Calcd for C20H17FN2O4: C,
65.21; H, 4.65; N, 7.60. Found: C, 64.97; H, 4.57; N, 7.48.
1
as yellow needles: mp 138-140 °C; H NMR (CDCl3) δ 1.04
(6H, d, J ) 6.6 Hz), 2.26 (1H, nonet, J ) 6.9 Hz), 2.75 (2H, d,
J ) 6.9 Hz), 3.69 (3H, s), 5.13 (2H, s), 7.15 (2H, t, J ) 8.7 Hz),
7.78 (1H, s), 8.23 (2H, dd, J ) 8.7, 5.7 Hz); 13C NMR (CDCl3)
δ 22.6, 26.5, 42.1, 58.5, 100.7, 116.2 (d, J ) 25.9 Hz), 130.1 (d,
J ) 3.9 Hz), 132.5 (d, J ) 3.2 Hz), 136.3 (d, J ) 10.6 Hz),
141.3 (d, J ) 1.7 Hz), 154.9, 158.4 (d, J ) 1.7 Hz), 160.5, 164.8
(d, J ) 306.1 Hz); HRESI-FTMS calcd for C17H20N2O4F (M +
H) 335.1401, found 335.1402. Anal. Calcd for C17H19FN2O4: C,
61.07; H, 5.73; N, 8.38. Found: C, 60.84; H, 5.77; N, 8.30.
Syn th esis of 1-Meth oxym eth oxy-3-isobu tyl-5-Z-m -
flu or oben zylid en e-3H-p yr a zin e-2,6-d ion e (23e). A similar
oxidation of PMB derivative 21e (475 mg, 1.08 mmol) with 4
equiv of DDQ and crystallization of the residue from methylene
chloride-hexane followed by acetone-hexane yielded (125 mg,
36%) desired product 23e as pale rosettes: mp 98-102 °C; 1H
NMR (CDCl3) δ 1.04 (6H, d, J ) 6.9 Hz), 2.28 (1H, nonet, J )
6.6 Hz), 2.76 (2H, d, J ) 6.9 Hz), 3.68 (3H, s), 5.12 (2H, s),
7.17 (1H, m), 7.40 (1H, dt, J ) 8.1, 6.0 Hz), 7.71 (1H, brd, J )
∼6.0 Hz), 7.75 (1H, s), 8.16 (1H, brd, J ) 7.2 Hz); 13C NMR
(CDCl3) δ 22.6, 26.4, 42.2, 58.5, 100.7, 118.9 (d, J ) 25.9 Hz),
119.7 (d, J ) 27.8 Hz), 130.1 (d, J ) 9.6 Hz), 133.7, 125.4 (d,
J ) 10.3 Hz), 140.8 (d, J ) 3.7 Hz), 154.8, 159.2, 160.3, 162.6
(d, J ) 193.9 Hz); HRESI-FTMS calcd for C17H20FN2O4 (M +
H) 335.1401, found 335.1399. Anal. Calcd for C17H19FN2O4: C,
61.07; H, 5.73; N, 8.38. Found: C, 60.76; H, 5.48; N, 8.20.
Syn th esis of 1-Meth oxym eth oxy-3-isobu tyl-5-Z-o-
flu or oben zylid en e-3H-p yr a zin e-2,6-d ion e (23f). A similar
oxidation of PMB derivative 21f (360 mg, 0.82 mmol) with
DDQ (0.75 g, 4 equiv) overnight and crystallization of the
residue from methylene chloride-hexane followed by acetone-
hexane furnished (50 mg, 19%) desired product 23f as fine pale
DDQ Oxid a tion of E-Isom er 22a . DDQ (202 mg, 3 equiv)
was added to a biphasic mixture of p-methoxybenzyl derivative
22a (120 mg, 0.30 mmol) at 0 °C in methylene chloride (5 mL)
and water (2.5 mL). After being stirred at 0 °C for 20 min, the
starting compound was consumed in favor of two major
products. The crude reaction mixture was filtered through a
small bed of Celite and silica gel and then purified by
preparative TLC developing in hexanes-EtOAc (4:1). Three
major bands were eluted to give 6 mg of 24 and 15 mg each of
compounds 26 and 27 all as gum. 26: 1H NMR (CDCl3) δ 0.84
(6H, d, J ) 6.3 Hz), 2.64 (1H, m), 3.70 (3H, s), 3.79 (3H, s),
4.90 (2H, s), 5.11 (2H, s), 5.73 (1H, d, J ) 11.4 Hz), 6.88 (2H,
d, J ) 8.7 Hz), 7.07 (2H, d, J ) 8.7 Hz); 13C NMR (CDCl3) δ
22.8, 26.2, 44.9, 55.3, 57.9, 101.1, 114.3, 123.6, 124.6, 127.2,
127.8, 151.7, 159.1; FABMS m/z 357 [M + Na]; HRESI-FTMS
calcd for C17H23N2O5 (M + H) 335.1601, found 335.1594. 27:
1H NMR (CDCl3) δ 0.97 (6H, d, J ) 6.6 Hz), 3.66 (1H, m), 3.70
(3H, s), 3.80 (3H, s), 4.68 (2H, s), 5.09 (2H, s), 5.28 (1H, d, J )
10.2 Hz), 6.87 (2H, d, J ) 8.7 Hz), 7.16 (2H, d J ) 8.7 Hz); 13
C
NMR (CDCl3) δ 22.9, 25.4, 43.1, 55.3, 57.8, 101.2, 114.2, 124.2,
127.1, 128.5, 129.1, 150.3, 158.2, 159.2; FABMS m/z 357 [M +
Na]; HRESI-FTMS calcd for C17H23N2O5 (M + H) 335.1601,
found 335.1594.
Syn th esis of 1-Hyd r oxy-3-isobu tyl-5-(2-m eth yl-Z-p r op -
1-en yl)-3H-p yr a zin e-2,6-d ion e (F lu tim id e, 1a ). To a cooled
(0 °C) solution of MOM ether 23a (6 mg, 0.025 mmol) in
anhydrous methylene chloride (1 mL) was added TFA (0.3 mL),
and the solution was stirred under nitrogen for 30 min followed
by at room temperature for 5 h. The progress of the reaction
was monitored by TLC (hexanes-EtOAc, 7:3) and reversed-
phase HPLC (Zorbax RX C-8, 4.6 × 250 mm, 50% aqueous
CH3CN +0.1%TFA at 40 °C at a flow rate of 1 mL/min). After
completion of the reaction the volatile materials were evapo-
rated under stream of nitrogen which gave almost pure (NMR
and HPLC) product. The product was purified on a same
analytical HPLC column to give pure product 1a (2 mg) as a
gum which was identical6a with the natural products in all
respect (UV, NMR, and HPLC).
In a similar reaction 190 mg of the MOM ether 23a was
deprotected and purified on a Zorbax RX C-8 (22 × 250 mm)
column and eluted with 50% aqueous CH3CN containing 0.1%
TFA at a flow rate of 8 mL/min to give the clean N-hydroxy
product (20 mg, 12.5%). It was difficult to improve the yield
due to hydrolysis of imine bond and other enamine reactions
in larger scale preparations.
1
needles: mp 86-90 °C; H NMR (CDCl3) δ 1.04 (6H, d, J )
6.6 Hz), 2.26 (1H, nonet, J ) 6.9 Hz), 2.75 (2H, d, J ) 7.2 Hz),
3.69 (3H, s), 5.13 (2H, s), 7.14 (1H, ddd, J ) 9.5, 8.4, 1.2 Hz),
7.23 (1H, t, J ) 7.2 Hz), 7.46 (1H, m), 8.16 (1H, s), 7.73 (1H,
dt, J ) 7.8 Hz); 13C NMR (CDCl3) δ 22.6, 26.5, 42.2, 58.5, 100.7,
115.7 (d, J ) 26.6 Hz), 121.9 (d, J ) 11.6 Hz), 124.5 (d, J )
4.3 Hz), 133.3 (d, J ) 9.3 Hz), 133.7 (d, J ) 11.1 Hz), 133.8 (d,
J ) 2.2 Hz), 134.0, 154.9, 159.1, 160.1, 162.7 (d, J ) 307.3
Hz); HRESI-FTMS calcd for C17H20FN2O4 (M + H) 335.1401,
found 335.1409. Anal. Calcd for C17H19FN2O4: C, 61.07; H,
5.73; N, 8.38. Found: C, 61.10; H, 5.61; N, 8.13.
Syn th esis of 1-Meth oxym eth oxy-3-ben zyl-5-(2-m eth yl-
Z-p r op -1-en yl)-3H-p yr a zin e-2,6-d ion e (23g). DDQ oxida-
tion of Z-olefin 21g (660 mg) and purification on silica gel gave
the desired imine 23g (40 mg) as an oil and 65 mg of
diketoamide 25 as a semisolid. 23g: 1H NMR (CDCl3) δ 1.12
(6H, d, J ) 6.9 Hz), 3.50 (1H, m), 3.64 (3H, s), 4.06 (2H, s),
5.06 (2H, s), 7.15 (1H, d, J ) 9.9 Hz), 7.24-7.36 (5H, m); 13C
NMR (CDCl3) δ 21.8, 27.5, 39.8, 58.4, 100.6, 126.9, 128.5, 129.5,
134.0, 135.7, 155.0, 156.4, 159.3, 159.7; HRESI-FTMS calcd
for C17H21N2O4 (M + H) 317.1495, found 317.1483. Anal. Calcd

Synthesis of Natural Flutimide
J . Org. Chem., Vol. 66, No. 16, 2001 5515
Syn th esis of 1-h yd r oxy-3-isobu tyl-5-Z-ben zylid en e-3H-
p yr a zin e-2,6-d ion e (1b). To a cooled (0 °C) solution of MOM
ether 23b (35 mg) in methylene chloride (2.0 mL) was added
TFA (0.5 mL), and the solution was stirred for 30 min under
nitrogen followed by 5 h at room temperature. Solvents were
removed under a stream of nitrogen and then dried under
vacuum overnight. The residue was crystallized from meth-
ylene chloride-hexane to give the N-hydroxy compound 1b
(25 mg, 83.3%) as a yellow powder: mp 138-140 °C; 1H NMR
(CDCl3) δ 1.05 (6H, d, J ) 6.6 Hz), 2.31 (1H, nonet J ) 6.6
Hz), 2.79 (2H, d, J ) 7.2 Hz), 7.48 (3H, m), 7.90 (1H, s), 8.23
(2H, dd, J ) 8.1, 1.8 Hz); 13C NMR (CDCl3) δ 22.6, 26.5, 42.1,
129.0, 131.6, 132.5, 133.5, 134.5, 144.2, 152.1, 157.4, 159.2;
HRESI-FTMS calcd for C15H17N2O3 (M + H) 273.1233, found
273.1244. Compound 1b was also produced upon deprotection
of 23g.
(2H, s), 6.83 (2H, d, J ) 9.3 Hz), 7.36 (5H, m), 7.81 (1H, s),
7.95 (2H, d, J ) 9.0 Hz); HRESI-FTMS calcd for C19H17N2O4
(M + H) 337.1183, found 337.1189.
Syn th esis of 1-Hyd r oxy-3-ben zyl-5-Z-p-flu or oben zyli-
d en e-3H -p yr a zin e-2,6-d ion e (2b ) a n d 1-H yd r oxy-3-p -
flu or oben zyl-5-Z-ben zylid en e-3H-p yr a zin e-2,6-d ion e (3).
Deprotection of the MOM ether 23i (50 mg) under the
conditions described above gave two isomeric products 2b and
3. After completion of the reaction, the products were purified
by reversed-phase HPLC on a Zorbax RX C-8 (45% aqueous
CH3CN containing 0.1% TFA at a flow rate of 1 mL/min). The
fractions were quickly lyophilized to give 4 mg of product 3
(tR ) 13.7 min) and 4 mg of product 2a (tR ) 14.7 min) as
amorphous solids. 2a : mp 138-142 °C; 1H NMR (CDCl3) δ
4.23 (2H, s), 6.98 (2H, t, J ) 8.8 Hz), 7.32-7.40 (5 Hz, m),
7.79 (1H, s), 7.93 (2H, dd, J ) 8.8, 5.6 Hz); CIMS m/z 325 [M
+ H]; HRESI-FTMS calcd for C18H14FN2O3 (M + H) 325.0983
Syn th esis of 1-Hyd r oxy-3-isobu tyl-5-Z-p-m eth oxyben z-
ylid en e-3H-p yr a zin e-2,6-d ion e (1c). TFA (0.3 mL) was
added to a cooled (0 °C) solution of MOM ether 23c (25 mg) in
methylene chloride (1.0 mL), and the solution was stirred for
30 min under nitrogen followed by overnight at room temper-
ature. The solvents were removed under a stream of nitrogen,
and the residue was triturated with CH3CN and filtered to
give 5 mg of product 1c as a yellow solid. The filtrate was
chromatographed on a Zorbax RX C-8 (9.4 × 250 mm) column
and eluted with 50% aqueous CH3CN at a flow rate of 4 mL/
min to give additional 4 mg of the product (total yield 41%):
1
found 325.0991. 3: mp 125-129 °C; H NMR (CDCl3) δ 4.20
(2H, s), 7.07 (2H, t, J ) 8.8 Hz), 7.31 (2H, dd, J ) 8.4, 5.2 Hz),
7.36 (2H, t, J ) 7.6 Hz), 7.48 (1H, dd, J ) 7.6, 1.2 Hz), 7.86
(1H, s), 7.94 (2H, dd, J ) 7.6, 1.2 Hz); CIMS m/z 325 [M + H];
HRESI-FTMS calcd for C18H14FN2O3 (M + H) 325.0983 found
325.0988.
Syn th esis of 1-Hydr oxy-3-isobu tyl-5-p-flu or oben zylpip-
er a zin e-2,6-d ion e (28). To a solution of imine 23d (6.6 mg)
in EtOAc (1 mL) and methanol (0.2 mL) was added 10% Pd/C
(5 mg), and the mixture was evacuated and flushed with
hydrogen. The evacuation and flush cycle was repeated three
times and was finally connected to a balloon filled with
hydrogen. The starting material was consumed within 20 min
(HPLC, Zorbax RX C-8, 50% aqueous CH3CN, 1 mL per min)
and a single product was formed. Catalyst was removed by
filtration and solvents were evaporated under reduced pres-
sure to give a gum, which was triturated with methylene
chloride-hexane to give a colorless solid (6.1 mg) of 28: mp
104-108 °C; 1H NMR (CDCl3) δ 0.86 (3H, d, J ) 6.3 Hz), 0.91
(3H, d, J ) 6.6 Hz), 1.46 (1H, ddd, J ) 14.1, 9.0, 5.1 Hz), 1.74
(1H, m), 1.96 (1H, ddd, J ) 13.5, 9.3, 2.6 Hz), 3.15 (1H, dd, J
) 14.4, 7.5 Hz), 3.36 (1H, dd, J ) 14.4, 4.2 Hz), 3.57 (1H, dd,
J ) 9.0, 3.6 Hz), 3.82 (1H, dd, J ) 7.2, 4.5 Hz), 7.02 (2H, t, J
) 8.7 Hz), 7.23 (2H, dd, J ) 8.7, 5.7 Hz), 13C NMR (CDCl3) δ
21.4, 23.1, 24.6, 35.4, 39.3, 57.3, 59.7, 115.8 (d, J ) 25.3 Hz),
131.1 (d, J ) 10 Hz), 131.6 (d, J ) 3.5 Hz), 162.1 (d, J ) 294.3
Hz), 167.5, 169.0; ESIMS m/z 295 [M + H], 589 (2M + H];
HRESI-FTMS calcd for C15H20FN2O3 (M + H) 295.1452 found
295.1455.
1
mp 152-156 °C; H NMR (CDCl3) δ 1.05 (6H, d, J ) 6.6 Hz),
2.30 (1H, nonet, J ) 6.6 Hz), 2.77 (2H, d, J ) 7.2 Hz), 3.91
(3H, s), 6.99 (2H, d, J ) 9.3 Hz), 7.86 (1H, s), 8.27 (2H, d, J )
8.7 Hz); FABMS m/z 315 (M + 2Li - H); HRESI-FTMS calcd
for C16H19N2O4 (M + H) 303.1339, found 303.1350.
Syn th esis of 1-Hyd r oxy-3-isobu tyl-5-Z-p-flu or oben z-
ylid en e-3H-p yr a zin e-2,6-d ion e (1d ). MOM ether 23d (60
mg) was deprotected with TFA and crystallized from methyl-
ene chloride-hexane to afford the N-hydroxy compound 1d (45
mg, 86.5%) as a yellow needles: mp 168-170 °C; 1H NMR
(CDCl3) δ 1.04 (6H, d, J ) 6.6 Hz), 2.28 (1H, nonet, J ) 6.9
Hz), 2.78 (2H, d, J ) 6.9 Hz), 7.26 (2H, t, J ) 9.0 Hz), 7.85
(1H, s), 8.27 (2H, dd, J ) 9.0, 5.7 Hz); 13C NMR (CDCl3) δ
22.6, 26.5, 42.1, 116.4 (d, J ) 26.1 Hz), 129.9 (d, J ) 3.9 Hz),
131.2 (d, J ) 3.2 Hz), 136.8 (d, J ) 10.9 Hz), 142.5, 152.3,
157.6 (d, J ) 1.6 Hz), 159.3, 165.1 (d, J ) 307 Hz); HRESI-
FTMS calcd for
291.1147. Anal. Calcd for C15H15FN2O3: C, 62.06; H, 5.21; N,
9.65. Found: C, 61.86; H, 4.99; N, 9.34.
C15H16FN2O3 (M + H) 291.1140, found
Syn th esis of 1-Hyd r oxy-3-isobu tyl-5-Z-m -flu or oben z-
ylid en e-3H-p yr a zin e-2,6-d ion e (1e). MOM ether 23e (60
mg) was deprotected with TFA and crystallized from acetone-
hexane to provide the N-hydroxy compound 1e (35 mg, 67.2%)
Syn t h esis of 3S-Isobu t yl-4-p -m et h oxyb en zyl-5S-[(p -
flu or op h en yl)-1R-h yd r oxym et h yl]p ip er a zin e-2,6-d ion e
(30). To a cooled (0 °C) solution of 18d (600 mg) in a mixture
of methanol (8 mL) and water (1.5 mL) were added concen-
trated sulfuric acid (0.3 mL) and concentrated hydrochloric
acid (0.3 mL), and the solution was stirred at room tempera-
ture for 96 h. The progress of the reaction was monitored on
an analytical HPLC using a Zorbax RX C-8 column and 50%
aqueous CH3CN containing 0.1% TFA. After the formation of
N-hydroxy compound was maximized, a small aliquot was
extracted with EtOAc, washed with water, dried over Na2SO4,
and chromatographed over a Zorbax RX C-8 HPLC column.
Elution with a 40-50% aqueous CH3CN (0.1% TFA) gradient
at a flow rate of 10 mL per min eluted 29 between 42 and 43
min which upon lyophilization gave pure 29 as a amorphous
1
as yellow needles: mp 133-137 °C; H NMR (CDCl3) δ 1.05
(6H, d, J ) 6.9 Hz), 2.30 (1H, nonet, J ) 6.9 Hz), 2.78 (2H, d,
J ) 7.2 Hz), 7.21 (1H, ddt, J ) 8.4, 2.7, 1.2 Hz), 7.43 (1H, dt,
J ) 8.1, 6.0 Hz), 7.77 (1H, d, J ) 7.8 Hz), 7.82 (1H, s), 8.18
(1H, dt, J ) 10.5, 2.2, 2.0 Hz); HRESI-FTMS calcd for C15H16
-
FN2O3 (M + H) 291.1140, found 291.1144. Anal. Calcd for
C
15H15FN2O3‚ 0.10 H2O: C, 61.68; H, 5.24; N, 9.59. Found: C,
61.46; H, 4.84; N, 9.56.
Syn th esis of 1-Hyd r oxy-3-isobu tyl-5-Z-o-flu or oben z-
ylid en e-3H-p yr a zin e-2,6-d ion e (1f). MOM ether 23f (20 mg)
was similarly deprotected with TFA and crystallized from
acetone-hexane to furnish the N-hydroxy compound 1f (10
mg, 57.6%) as yellow needles: mp 126-130 °C; 1H NMR
(CDCl3) δ 1.04 (6H, d, J ) 6.6 Hz), 2.30 (1H, nonet, J ) 6.9
Hz), 2.79 (2H, d, J ) 7.2 Hz), 7.16 (1H, ddd, J ) 9.5, 8.4, 1.2
Hz), 7.25 (1H, t, J ) 5.1 Hz), 7.50 (1H, m), 8.24 (1H, s), 8.78
(1H, dt, J ) 7.8, 1.8 Hz); HRESI-FTMS calcd for C15H16FN2O3
(M + H) 291.1140, found 291.1146.
Syn th esis of 1-Hyd r oxy-3-ben zyl-5-Z-p-m eth oxyben z-
ylid en e-3H-p yr a zin e-2,6-d ion e (2a ). Deprotection of the
MOM group from MOM ether 23h (60 mg) with TFA followed
by preparative HPLC on a Zorbax RX C-8 (22 × 250 mm)
column and elution with 50% aqueous CH3CN at a flow rate
of 10 mL/min gave 15 mg (25%) of pure product 2a as a yellow
powder: mp 150-152 °C; 1H NMR (CDCl3) δ 3.89 (3H, s), 4.12
1
powder: mp 66-70 °C; H NMR (CDCl3) δ 0.47 (3H, d, J )
6.0 Hz), 0.85 (3H, d, J ) 6.3 Hz), 1.50 (2H, m), 1.75 (1H, m),
3.20 (1H, d, J ) 12.9 Hz), 3.64 (1H, dd, J ) 10.2, 4.5 Hz), 3.70
(3H, s), 3.86 (1H, d, J ) 12.9 Hz), 4.10 (1H, d, J ) 7.5 Hz),
5.28 (1H, d, J ) 7.8 Hz), 6.65 (2H, d, J ) 8.7 Hz), 6.72 (2H, d,
J ) 8.7 Hz), 7.10 (2H, t, J ) 8.7 Hz), 7.33 (2H, dd, J ) 8.7, 5.4
Hz); ESIMS m/z 431 [M + H], 861 [2M + H]; HRESI-FTMS
calcd for C23H28FN2O5 (M + H) 431.1977, found 431.1977.
Titanium trichloride (460 mg) was added, and the remainder
of the reaction was stirred for an additional 16 h. Aqueous
NaHCO3 was added to the reaction mixture, and the product
was extracted with EtOAc, which was washed with water,
dried over Na2SO4, evaporated under reduced pressure, and
chromatographed on a silica gel column and eluted with 30-

5516 J . Org. Chem., Vol. 66, No. 16, 2001
Singh and Tomassini
40% EtOAc in hexane to give pure 30 (150 mg) as an
amorphous powder: mp 58-60 °C; 1H NMR (CDCl3) δ 0.43
(3H, d, J ) 6.0 Hz), 0.83 (3H, d, J ) 6.0 Hz), 1.42 (2H, m),
1.70 (1H, m), 3.18 (1H, d, J ) 12.9 Hz), 3.40 (1H, dd, J ) 10.8,
4.2 Hz), 3.76 (3H, s), 3.85 (1H, d, J ) 12.9 Hz), 3.94 (1H, d, J
) 8.1 Hz), 5.23 (1H, d, J ) 8.1 Hz), 6.65 (2H, d, J ) 9.0 Hz),
6.72 (2H, d, J ) 8.7 Hz), 7.10 (2H, t, J ) 8.7 Hz), 7.35 (2H, dd,
J ) 8.7, 5.4 Hz), 8.19 (1H, brs, NH); 13C NMR (CDCl3) δ 20.8,
23.2, 23.8, 37.0, 52.3, 55.3, 58.8, 62.5, 72.0, 113.9, 115.4 (d, J
) 25.7 Hz), 127.8, 129.3 (d, J ) 10 Hz), 130.2, 135.1 (d, J )
3.9 Hz), 159.3, 162.8 (d, J ) 294.8 Hz), 173.1, 173.5; ESIMS
m/z 415 [M + H], 829 [2M + H]; HRESI-FTMS calcd for
25%]; HRESI-FTMS calcd for C23H26FN2O3 (M + H) 397.1922,
found 397.1919.
Syn th esis of 3-Isobu tyl-5-Z-p-flu or oben zylid en e-3H-
p yr a zin e-2,6-d ion e (32). DDQ oxidation of 31 (80 mg) using
the conditions described earlier followed by workup and
crystallization from acetone-hexane gave 20 mg of imine
product 32 as yellow needles: mp 168-172 °C; 1H NMR
(CDCl3) δ 1.05 (6H, d, J ) 6.9 Hz), 2.27 (1H, m), 2.74 (2H, d,
J ) 6.9 Hz), 7.16 (2H, t, J ) 8.7 Hz), 7.75 (1H, s), 8.22 (2H,
dd, J ) 8.7, 5.4 Hz); EIMS m/z 274 [M, 50%]; HRESI-FTMS
calcd for C15H16FN2O2 (M + H) 275.1190, found 275.1180.
C
23H28FN2O4 (M + H) 415.2027, found 415.2024.
Ack n ow led gm en t. The authors are grateful to Drs.
J erry Liesch, Debbie Zink, and Dr. Z. Guan for mass
spectral support, Dr. Michael Goetz for encouragement,
and especially Mr. K. Herath for considerable technical
support.
Syn t h esis of 3S-Ben zyl-4-p -m et h oxyb en zyl-5-Z-p -
flu or oben zylid en ep ip er a zin e-2,6-d ion e (31). The elimina-
tion reaction was repeated on 150 mg of the substrate 30 using
the conditions of aromatic series and a similar chromatography
on silica gel employing 5-15% EtOAc in hexane gave elimina-
tion product 31 (90 mg, 63%) as a yellow solid: mp 50-55 °C;
1H NMR (CDCl3) δ 0.84 (3H, d, J ) 6.6 Hz), 0.87 (3H, d, J )
6.6 Hz), 1.45 (1H, ddd, J ) 14.1, 9.0, 5.4 Hz), 1.60 (1H, ddd, J
) 13.8, 9.9, 5.4 Hz), 1.81 (1H, m), 3.64 (1H, dd, J ) 9.6, 5.1
Hz), 3.78 (3H, s), 3.92 (2H, ABq, J ) 13.5 Hz), 6.82 (2H, d, J
) 9.0 Hz), 7.10 (2H, d, J ) 8.7 Hz), 7.15 (2H, t, J ) 8.7 Hz),
7.52 (1H, s), 8.00 (2H, dd, J ) 8.7, 5.4 Hz); EIMS m/z 396 [M,
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 1b,c,f, 2a , 3, 17a , 21a , 23g, 24, 26, 27, and 29-
32. This material is available free of charge via the Internet
at http://pubs.acs.org.
J O015665D