Synthesis and in vitro evaluation of new 8-amino-1,4-benzoxazine derivatives as neuroprotective antioxidants

Article abstract of DOI:10.1021/jm991105j

A series of new 8-amino-1,4-benzoxazine derivatives 5a-o was synthesized and examined for their intrinsic cytotoxicity and their capacity to inhibit oxidative stress-mediated neuronal degeneration in neuronal cell cultures. In particular, substituent effects at the 3- and 8-positions of the 1,4- benzoxazine ring were investigated by in vitro evaluation. In this aim, 3- alkyl substituents seemed to be essential for efficient neuroprotective activity. Furthermore, within the subseries of substituted 3-alkyl benzoxazines, the most active derivatives were those bearing an 8-benzylamino substituent. From the combined results of both toxicity and neuroprotection expressed in terms of the safety index, 8-benzylamino-substituted-3-alkyl- 1,4-benzoxazines were identified as the most promising compounds, owing to their potent neuroprotective activity without the manifestation of intrinsic cytotoxicity.

Full text of DOI:10.1021/jm991105j

J . Med. Chem. 1999, 42, 5043-5052
5043
Syn th esis a n d in Vitr o Eva lu a tion of New 8-Am in o-1,4-ben zoxa zin e Der iva tives
a s Neu r op r otective An tioxid a n ts
Martine Largeron,† Brian Lockhart,‡ Bruno Pfeiffer,§ and Maurice-Bernard Fleury*,†
Laboratoire de Chimie Analytique et Electrochimie, UMR 8638 CNRS - Universite´ Rene´ Descartes, Faculte´ des Sciences
Pharmaceutiques et Biologiques, 4 avenue de l’Observatoire, 75270 Paris Cedex 06, France, Institut de Recherche Servier,
125 chemin de Ronde, 78290 Croissy-sur-Seine, France, and ADIR, 1 rue Carle He´bert, F -92415 Courbevoie Cedex, France
Received J une 22, 1999
A series of new 8-amino-1,4-benzoxazine derivatives 5a -o was synthesized and examined for
their intrinsic cytotoxicity and their capacity to inhibit oxidative stress-mediated neuronal
degeneration in neuronal cell cultures. In particular, substituent effects at the 3- and 8-positions
of the 1,4-benzoxazine ring were investigated by in vitro evaluation. In this aim, 3-alkyl
substituents seemed to be essential for efficient neuroprotective activity. Furthermore, within
the subseries of substituted 3-alkyl benzoxazines, the most active derivatives were those bearing
an 8-benzylamino substituent. From the combined results of both toxicity and neuroprotection
expressed in terms of the safety index, 8-benzylamino-substituted-3-alkyl-1,4-benzoxazines were
identified as the most promising compounds, owing to their potent neuroprotective activity
without the manifestation of intrinsic cytotoxicity.
Reactive oxygen species are byproducts of normal
metabolic processes in aerobic environments.1 These
species can exert cytotoxicity by damaging critical
cellular components necessary for viability.2 Examples
of the reactive oxygen species are superoxide, hydrogen
peroxide, and hydroxyl radicals produced by sources
such as bioreductively activated xenobiotics or by low-
molecular-weight complexes of transition metals such
as iron and copper via the Fenton reaction.
ronal oxygen and glucose metabolism, as well as an-
tagonism of aminergic neurotransmission impairment
induced by temporary ischemia.6,7 Furthermore, exifone
demonstrated remarkable scavenger properties against
free radicals.8 Unfortunately, after reports of severe
hepatotoxicity associated with the product, in 1990, the
registration was revoked and exifone was withdrawn
from the market.
On the basis of previous results concerning the
possible role of highly electrophilic oxidation products
(quinone or iminoquinone) in the toxicological effects of
drugs,9-11 we hypothesized that a potential toxic me-
tabolite of exifone could be the transient reactive
o-quinone species, which would bind irreversibly to the
NH2 and SH residues of cellular proteins to form
conjugates.12-14 Our objective was then to scavenge the
transient reactive o-quinone by the formation of an
adduct blocking the electrophilic sites generally at-
tacked by NH2 and SH membrane residues and conse-
quently to prevent toxicity. A few years ago, we de-
scribed the first electrochemical and chemical syntheses
of novel 1,4-benzoxazine derivatives I and II structur-
ally related to exifone (Chart 1) that have been identi-
fied as efficient antioxidants and significantly less
hepatotoxic than exifone.15,16
Elimination of these oxidants is provided by enzy-
matic and nonenzymatic mechanisms. Enzymatic anti-
oxidants include superoxide dismutase, catalase, and
glutathione peroxidase which catalytically detoxify oxi-
dants, whereas nonenzymatic antioxidants are prima-
rily reducing agents, such as vitamin C, vitamin E, and
glutathione, which can scavenge oxidants by hydrogen
atom donation in a stoichiometric manner.3 Imbalances
in the detoxification of reactive oxygen species relative
to their production lead to what is a widely accepted
phenomenon called “oxidative stress”. In most cases, the
defense provided by the enzymatic and nonenzymatic
antioxidants is adequate. However, in acute situations,
such as cerebral ischemia, an increased “oxidative load”
results in “oxidative stress”, culminating in accelerated
neurodegenerative mechanisms. In addition, oxidative
stress may play a role in the progression of many
neurodegenerative pathologies of the central nervous
system, including Parkinson’s and Alzheimer’s disease.
Consequently, supplementation with exogenous anti-
oxidants could represent an important therapeutic
potential to minimize central nervous system damage
and has prompted the search for such substances.
In this context, exifone (Adlone) was launched in
France, in 1988, for the treatment of cognitive problems
in the elderly4,5 and was reported to have a number of
pharmacological properties including activation of neu-
To continue these investigations, we describe herein
the electrochemical and chemical syntheses of a new set
of 1,4-benzoxazine derivatives 5a -o bearing a 8-amino
substituent (Chart 1). Preliminary in vitro assays of
these molecules have been performed in order to explore
the structural requirements for efficient antioxidative
activity, without the manifestation of intrinsic cytotox-
icity.
Ch em istr y
General chemical methods currently available so far
for the preparation of 1,4-benzoxazines17-21 were not
adapted to the synthesis of a series of 8-amino-1,4-
benzoxazine derivatives for a rapid in vitro biological
† UMR 8638 CNRS - Universite´ Rene´ Descartes.
‡ Institut de Recherche Servier.
§ ADIR.
10.1021/jm991105j CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/13/1999

5044 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Largeron et al.
Ch a r t 1
electrochemical oxidation. Nevertheless, this procedure
gave lower yields of 1,4-benzoxazin-8-one derivatives
3a -c ranging from 35 to 60% (Table 1, method B).
Reduction of the carbonyl function at the C-8 position
of 1,4-benzoxazin-8-one derivatives could be achieved
using zinc in methanolic acetic acid solution, leading to
the phenolic derivatives 6a -c in roughly 60% yields
(Table 1, method G).
Particular difficulty was noted with the chemical
synthesis of 8-amino-1,4-benzoxazines 5a-o which failed,
in several cases, due to the instability of the Schiff base
intermediate 4. If the latter was sufficiently stable, it
could be reduced using zinc in methanolic acetic acid
solution at the end of the reaction with the excess of
amine R3-NH2, giving the expected 8-amino derivatives
(Table 2, method E). Conversely, when the Schiff base
4 was unstable, the electrochemical procedure (Table
2, method D) was preferred over the chemical one since
the Schiff base 4 could be reduced as soon as it appeared
in the electrolysis solution. All targeted compounds are
detailed in Tables 1 and 2.
Resu lts a n d Discu ssion
The intrinsic neurotoxicity as well as the neuropro-
tective activity of 1,4-benzoxazines were assessed in
vitro on murine HT-22 hippocampal cell cultures and
compared with those of parent exifone. The results are
presented in Tables 3 and 4.
evaluation. These procedures would require, as the
starting materials, polysubstituted o-aminophenols which
are difficult to prepare. Consequently, it is for these
reasons that we have recently developed a convenient
general two-step one-pot electrochemical procedure for
the synthesis of 8-amino-1,4-benzoxazine derivatives.22
The pathway involved the initial oxidation of (3,4-
dihydroxy-2-methoxyphenyl)(phenyl) methanone 1, lead-
ing to the formation of the transient 3,4-quinone 2
(Scheme 1). The subsequent step consisted of a substi-
tution reaction of the 2-methoxy group by an amino-
alcohol [CH2OH-C(R1,R2)-NH2], affording, after intramo-
lecular ring closure, the 1,4-benzoxazin-8-one derivatives
3a -c which could be isolated in good yields ranging
from 65 to 95% (Table 1, method A). However, the
electrochemical procedure could be pursued without
isolation of the latter that was allowed to react with an
excess of amine R3-NH2, producing the Schiff base
intermediate 4. This intermediate was subsequently
reduced through a two-electron transfer to 8-amino
derivatives 5a -o in moderate yields ranging from 15
to 60% (Table 2, method C).
To explore the influence of the amino chain at the C-8
position of the benzoxazine ring, the synthesis of 8-hy-
droxy derivatives 6a -c was targeted from the corre-
sponding 1,4-benzoxazin-8-ones 3a-c. The electrochemi-
cal procedure involved a two-electron reduction of the
carbonyl function at the C-8 position, producing, after
elimination of one molecule of water, the phenolic
derivatives 6a -c in roughly 80% yields (Table 1, method
F).
To estimate potential pro-oxidant effects23 and to
select a concentration range lacking intrinsic toxicity,
suitable for studying the neuroprotective activity of the
compound, the intrinsic neurotoxic effects of each
compound was evaluated following two different meth-
ods. Neurotoxicity was monitored either by reduction
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT reduction assay), which allows an evalu-
ation of the “redox state” of the cells and emphazises
oxidative stress, or by quantification of cellular lysis
(death) after measurement of the lactate dehydrogenase
(LDH) activity released from damaged cells into the
culture supernatant. The maximum tolerated concen-
tration (MTC) and the concentration producing 50%
toxicity (TC50) were estimated for each tested compound
using both MTT and LDH determinations.
Neuroprotective properties of 1,4-benzoxazine deriva-
tives were estimated through their protective effects
against L-homocysteic acid (L-HCA) cytotoxicity. Previ-
ous cell culture toxicity studies have demonstrated that
depletion of intracellular glutathione levels by the
competitive inhibition of cystine uptake, via the cystine/
glutamate antiporter Xc-, and by exposure of immature
primary neurons or certain neuronal cell lines to L-HCA
results in oxidative stress mediated neuronal degenera-
tion which was attenuated with antioxidants.24,25 The
concentration producing 50% protection (PC50) was
estimated for both MTT and LDH determinations. On
the basis of these results, the MTC/PC50 ratio was
calculated as an index of the pro- and antioxidant effects
of each compound and was termed the “safety index”.
As reported in Tables 3 and 4, the MTC determined
from the MTT reduction assay appeared to be the most
sensitive parameter for all evaluated compounds. With
regard to this parameter, all targeted 1,4-benzoxazin-
8-one derivatives 3a -c as well as their 8-hydroxy
Last, in order to prepare the optimal candidate at the
preparative scale for further in vivo evaluation, we
looked for a chemical extrapolation of our electrochemi-
cal procedure. In agreement with the potential value
used in the course of the controlled anodic potential
electrolysis, Ag2O was selected as a surrogate of the

Synthesis of 8-Amino-1,4-benzoxazine Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5045
Sch em e 1a
a
Reagents and conditions: (a) MeOH, Ag2O, or electrochemical oxidation at a platinum anode (E ) +0.4 V vs sce), rt; (b) MeOH,
CH2OH-C (R1, R2)-NH2, rt; (c) MeOH, R3-NH2, rt; (d) MeOH/AcOH, zinc, or electrochemical reduction at a mercury cathode (E ) -1.0 V
vs sce), rt; (e) MeOH/AcOH, zinc, or electrochemical reduction at a mercury cathode (E ) -1.2 V vs sce), rt.
Ta ble 1. Physical Properties of 2H-1,4-Benzoxazin-8-ones 3 and 2H-1,4-Benzoxazin-5-yl-(phenyl)-methanones 6
a
b
Procedure is described in the Experimental Section. Crystallized following concentration of the flash column chromatography solvent.
derivatives 6a -c demonstrated MTC values of the same
order of magnitude as the PC50 values, as indicated by
the low safety index values. Similarly, with exifone, we
were unable to determine, in our tests, a concentration
range for which antioxidant effects could be achieved
without the manifestation of intrinsic toxicity (compare
PC50 with MTC).
sults in vitro with, in most cases, very significant safety
index values (up to 71 for compound 5d ).
First, apart from compounds 5c, 5e, and 5i (which
bear an 8-isopentylamino or an 8-isobutylamino sub-
stituent), introduction of an amino chain at the 8-posi-
tion of the benzoxazine ring induced a significant
decrease in in vitro toxicity when compared with both
the 8-hydroxy analogues 6a -c and exifone: compare
compounds 5a and 5b (MTC ) 100 µM) with their
Contrary to compounds 3 and 6, the new series of
8-amino-1,4-benzoxazines 5a -o showed promising re-

5046 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Largeron et al.
Ta ble 2. Physical Properties of 2H-1,4-Benzoxazin-5-yl-(phenyl)-methanones 5
a
b
Procedure is described in the Experimental Section Crystallized after cooling to -40 °C. Abbrevations: tert-butyl (But), isopentyl
(Pei), isobutyl (Bui), benzyl (Bzl).
Ta ble 3. In Vitro Neuroprotective Activity of 2H-1,4-Benzoxazin-8-ones 3 and 2H-1,4-Benzoxazin-5-yl-(phenyl)-methanones 6
a
In vitro neurotoxicity monitored either by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reduction
assay) or by quantification of cellular lysis after measurement of the lactate dehydrogenase (LDH) activity released from damaged cells
b
into the culture supernatant. In vitro neuroprotective activity estimated through their protective effects against L-homocysteic acid
(L-HCA) cytotoxicity. Abbreviations: MTC, maximum tolerated concentration; TC50, concentration producing 50% toxicity; PC50
concentration producing 50% protection.
,
8-hydroxy counterpart 6a (MTC ) 1 µM); similarly,
compare 5h (MTC > 250 µM) with 6b (MTC ) 25 µM).
From the data collected in Table 4, it could be concluded
that substituted 8-benzylamino, 8-phenylethylamino,

Synthesis of 8-Amino-1,4-benzoxazine Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5047
Ta ble 4. In Vitro Neuroprotective Activity of 2H-1,4-Benzoxazin-5-yl-(phenyl)-methanones 5
a
In vitro neurotoxicity monitored either by reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reduction
assay), or by quantification of cellular lysis after measurement of the lactate dehydrogenase (LDH) activity released from damaged cells
b
into the culture supernatant. In vitro neuroprotective activity estimated through their protective effects against L-homocysteic acid
(L-HCA) cytotoxicity. Abbreviations: MTC, maximum tolerated concentration; TC50, concentration producing 50% toxicity; PC50
concentration producing 50% protection.
,
and 8-hydroxyalkylamino benzoxazines exhibited a low
toxicity with relatively high MTC values whereas,
exclusive of compound 5k , substituted 8-isobutylamino
and 8-isopentylamino derivatives 5c, 5e, and 5i were
rather toxic. The nature of the substituents at the
3-position was apparently not critical for toxicity, since
compounds 5g, 5l, and 5n , all possessing an 8-[2-
hydroxy-1-(hydroxymethyl)ethyl] amino substituent,
showed the same MTC value (100 µM). Similar observa-
tions were made for compounds 5a , 5d , and 5h , with
an 8-benzylamino substituent.
Second, concerning the structure-activity relation-
ships within the series of 8-amino-1,4-benzoxazine
derivatives 5a -o, the results reported in Table 4
showed that the requirements for the substitutions on
both the 3- and 8-positions of the benzoxazine ring
might be quite stringent. The 3-alkyl substituents
seemed to be essential for efficient neuroprotective
activity, since 3,3-diphenyl derivatives 5j and 5k or
3-hydroxyalkyl derivatives 5l-o were clearly inactive.
Furthermore, within the subseries of substituted 3-alkyl
benzoxazines 5a -i, the most active derivatives are those
bearing an 8-benzylamino substituent (compounds 5a ,
5d , and 5h ) and, to a lesser extent, those bearing an
8-phenylethylamino substituent (compound 5b) or an
8-alkylamino group (compounds 5c, 5e, and 5i). Inter-
estingly, replacement of the 8-alkylamino by an 8-hy-
droxyalkylamino substituent resulted in a large de-
crease in activity (at least 5 times). So, it could be
concluded that alcoholic substituents abolished neuro-
protective activity, whatever their position (at C-3 or
C-8) on the 1,4-benzoxazine ring.
As mentioned above, the results of both toxicity and
activity were combined to determine a safety index
defined as MTC/PC50. The latter, reported in Table 4,
was used to estimate the therapeutic potential of
compounds 5a -o. It rapidly appeared that compounds
bearing 8-benzylamino as well as 3-alkyl substituents
exhibited the highest safety index. Consequently,
8-amino-1,4-benzoxazine derivatives 5a , 5d , and 5h ,
with safety index of 27, >71, and >36, respectively, were
considered as the most attractive compounds from our
in vitro evaluation.
Con clu sion s
We have synthesized and evaluated a series of 1,4-
benzoxazine derivatives as neuroprotective agents. Ow-
ing to their very low safety index, 1,4-benzoxazin-8-ones
3a -c and 8-hydroxy-1,4-benzoxazines 6a -c were not
considered as promising compounds. It is possible that
the 1,4-benzoxazin-8-one derivatives 3a -c could un-
dergo an opening reaction up to the 1,4-oxazine ring, to
generate the putative toxic 3,4-quinone species. If such
a species is the potential toxic metabolite of exifone as
suggested in the Introduction, it was not surprising that
1,4-benzoxazin-8-one derivatives 3a -c exhibited a po-

5048 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Largeron et al.
Dia ster eoisom er A: 1H NMR [300 MHz, (CD3)2SO] δ 1.00
(s, 9H, Me, But), 3.65 (m, 1H, 3-H), 4.05 (m, 1H, 2-CH2), 4.40
(m, 1H, 2-CH2), 5.25 (d, 1H, J ) 9 Hz, 7-H), 7.25 (d, 1H, J )
9 Hz, 6-H), 7.50 (m, 5H, aromatic, Ph), 8.25 (s, 1H, OH, D2O
exchanged), 12.60 (s, 1H, NH, D2O exchanged); 13C NMR [75
MHz, (CD3)2SO] δ 26.6 (Me, But), 35.1 (CQ, But), 56.8 (C-3),
58.2 (C-2), 86.7 (C-8a), 99.5 (C-5), 109.3 (C-7), 128.8, 129.4 and
131.3 (CH, Ph), 140.3 (CQ, Ph), 147.6 (C-6), 168.9 (C-4a), 190.9
(C-8), 194.0 (CO, 5-benzoyl).
Dia ster eoisom er B: 1H NMR [300 MHz, (CD3)2SO] δ 1.00
(s, 9H, Me, But), 3.55 (m, 1H, 3-H), 3.95 (m, 1H, 2-CH2), 4.30
(m, 1H, 2-CH2), 5.15 (d, 1H, J ) 9 Hz, 7-H), 7.15 (d, 1H, J )
9 Hz, 6-H), 7.50 (m, 5H, aromatic, Ph), 8.20 (s, 1H, OH, D2O
exchanged), 12.70 (s, 1H, NH, D2O exchanged); 13C NMR [75
MHz, (CD3)2SO] δ 26.2 (Me, But), 33.6 (CQ, But), 58.1 (C-2),
58.5 (C-3), 87.2 (C-8a), 99.8 (C-5), 109.1 (C-7), 128.8, 129.4 and
131.3 (CH, Ph), 140.3 (CQ, Ph), 147.1 (C-6), 169.6 (C-4a), 191.5
(C-8), 193.8 (CO, 5-benzoyl); MS (mixture of A and B) (DCI)
m/z ) 328 (MH+). Anal. (C19H21NO4) C, H, N.
Meth od B. To a solution of compound 1 (244 mg, 1 mmol)
and S-tert-leucinol (597 mg, 5 mmol) in methanol (500 mL)
was added Ag2O (575 mg, 5 mmol). The mixture was stirred
at room temperature for 2 h, under nitrogen. Then, the solid
residue was removed by filtration, and the filtrate was poured
into a molar acetic acid buffered aqueous solution of pH ∼ 4.5
(100 mL). The resulting aqueous alcoholic solution was treated
according to method A and afforded compound 3a (196 mg,
60%).
tent toxicity. Introduction of an amino chain at the
8-position of the 1,4-benzoxazine ring appeared to
constitute a method of detoxification, except for alkyl
amino chains such as isopentyl or isobutyl. However,
the real role of the 8-substituent is still unclear so far.
On the basis of structure-activity relationships within
the 8-amino-1,4-benzoxazine series, the 3-alkyl func-
tionality appeared to be essential for activity and we
can conclude that the most suitable substituents to date
are alkyl chains such as tert-butyl, methyl, or cyclopen-
tyl. Since the 3-substituents play an important role on
the intensity of the intramolecular hydrogen bonding
that has been evidenced between the oxygen of the
carbonyl group of the 5-benzoyl substituent and the
N-H group of the 1,4-benzoxazine ring, it is also
possible that some of these compounds may act in vivo
via the formation of chelates with metals such as copper
or iron. Additional structural biological studies (X-ray,
for example) would be necessary to provide fresh insight
into the structure-activity relationships within the
8-amino-1,4-benzoxazines series. These studies, as well
as efforts to examine the ability of these molecules to
afford chelates, are underway in our laboratories.
Finally, this study has culminated in the selection of
8-benzylamino-substituted-3-alkyl-1,4-benzoxazines with
greater neuroprotective activity, without the toxicity
exhibited by exifone.
Compounds 3b and 3c were prepared following a procedure
analogous to method A or method B (see Table 1).
5-Ben zoyl-3-sp ir o-1′-cyclop en t yl-8a -h yd r oxy-3,4,8,8a -
tetr a h yd r o-2H-1,4-ben zoxa zin -8-on e 3b: 1H NMR [300
MHz, (CD3)2SO] δ 1.70 to 2.10 (m, 8H, CH2, cyclopentyl), 3.85
(d, 1H, J ) 12 Hz, 2-CH2), 4.30 (d, 1H, J ) 12 Hz, 2-CH2),
5.20 (d, 1H, J ) 10 Hz, 7-H), 7.20 (d, 1H, J ) 10 Hz, 6-H),
7.50 (m, 5H, aromatic, Ph), 8.35 (s, 1H, OH, D2O exchanged),
12.5 (s, 1H, NH, D2O exchanged); 13C NMR [75 MHz, (CD3)2-
SO] δ 23.8, 24.2, 36.7 and 40.7 (CH2, cyclopentyl), 61.8 (C-3),
65.0 (C-2), 87.9 (C-8a), 99.7 (C-5), 108.9 (C-7), 128.7, 129.5 and
131.3 (CH, Ph), 140.4 (CQ, Ph), 147.1 (C-6), 167.8 (C-4a), 191.2
(C-8), 193.8 (CO, 5-benzoyl); MS (DCI): m/z ) 326 (MH+). Anal.
(C19H19NO4) C, H, N.
(R,S)-5-Ben zoyl-3-h yd r oxym eth yl-8a -h yd r oxy-3,4,8,8a -
tetr a h yd r o-2H-1,4-ben zoxa zin -8-on e 3c. Dia ster eoisom er
A: 1H NMR [300 MHz, (CD3)2SO] δ 3.40 to 3.90 (m, 3H, 3-CH2
and 3-H), 4.10 (m, 1H, 2-CH2), 4.40 (dd, 1H, J ) 6 Hz, J ) 12
Hz, 2-CH2), 5.20 (d, 1H, J ) 10 Hz, 7-H), 5.40 (s, 1H, 3-CH2OH,
D2O exchanged), 7.15 (d, 1H, J ) 10 Hz, 6-H), 7.50 (m, 5H,
aromatic, Ph), 8.30 (s, 1H, 8a-OH, D2O exchanged), 12.30 (s,
1H, NH, D2O exchanged); 13C NMR [75 MHz, (CD3)2SO] δ 50.9
(C-3), 58.2 (C-2), 62.8 (CH2OH), 87.5 (C-8a), 99.8 (C-5), 108.8
(C-7), 128.7, 129.3 and 130.4 (CH, aromatic, Ph), 140.6 (CQ,
Ph), 147.7 (C-6), 191.9 (C-8), 193.8 (CO, 5-benzoyl).
Exp er im en ta l Section
Ch em istr y. The syntheses of the compounds are described
by way of typical examples: the methods are denoted in the
same manner as in Tables 1 and 2. Melting points were
1
determined on a Ko¨fler block and were uncorrected. H NMR
and 13C NMR spectra were recorded on a Brucker AC 300
spectrometer operating at 300 MHz for 1H observations.
Chemical shifts (in ppm) are relative to internal tetrameth-
ylsilane. The measurements were carried out using the
standard pulse sequences. The carbon type (methyl, methyl-
ene, methide, or quaternary) was determined by DEPT experi-
ments. Mass spectra were recorded on a Nermag R 10-10C
spectrometer equipped with desorption chemical ionization
mode (DCI/NH3). All compounds were analyzed for C, H, and
N. Analytical results were within (0.4% of the theoretical
values unless otherwise indicated. Merck 60 (40-60 µm) and
Merck 60 F254 silica gel were used for column chromatography
and thin-layer chromatography, respectively. All reagents and
solvents were of commercial quality or were purified before
use.
Dia ster eoisom er B: 1H NMR [300 MHz, (CD3)2SO] δ 3.40
to 3.90 (m, 3H, 3-CH2 and 3-H), 3.80 (m, 1H, 2-CH2), 4.10 (m,
1H, 2-CH2), 5.15 (d, 1H, J ) 10 Hz, 7-H), 5.40 (s, 1H, OH,
3-CH2OH, D2O exchanged), 7.25 (d, 1H, J ) 10 Hz, 6-H), 7.50
(m, 5H, aromatic, Ph), 8.20 (s, 1H, 8a-OH, D2O exchanged),
12.20 (s, 1H, NH, D2O exchanged); 13C NMR [75 MHz, (CD3)2-
SO] δ 52.3 (C-3), 58.4 (C-2), 61.7 (CH2OH), 87.8 (C-8a), 99.8
(C-5), 108.9 (C-7), 128.7, 129.3 and 130.4 (CH, aromatic, Ph),
140.6 (CQ, Ph), 147.3 (C-6), 191.9 (C-8), 193.9 (CO, 5-benzoyl);
Electr och em istr y. The apparatus, cells, and electrodes
were identical with those described previously.26
Met h od A. (R,S)-5-Ben zoyl-3-ter t-b u t yl-8a -h yd r oxy-
3,4,8,8a -tetr a h yd r o-2H-1,4-ben zoxa zin -8-on e 3a . A solu-
tion of compound 1 (244 mg, 1 mmol), tetraethylammonium
perchlorate (TEAP) (2.3 g, 10 mmol), and S-tert-leucinol (597
mg, 5 mmol) in methanol (500 mL) was oxidized, under
nitrogen, at room temperature, at a platinum electrode [E )
+ 0.4 V vs saturated calomel electrode (sce)]. After exhaustive
oxidation, i.e., when a steady-state minimum value of the
current was recorded, the resulting solution was poured into
a molar acetic acid buffered aqueous solution of pH ∼ 4.5 (100
mL). The resulting hydroalcoholic solution was concentrated
to 100 mL, under reduced pressure, at 40 °C, and extracted
with ethyl acetate (200 mL). The organic phase was dried
(MgSO4) and evaporated. The residue was chromatographed
on silica with toluene-acetone (98:2) as the eluent, leading to
compound 3a (310 mg, 95%) as an amorphous yellow solid:
mp 185-187 (decomp). The latter contained a mixture (60:
40) of two diastereoisomers A and B that could not be
separated.
MS (mixture of A and B) (DCI) m/z ) 302 (MH+). Anal. (C16H15
-
NO5) C, H, N.
Meth od C. (R,S)-[8-Ben zyla m in o-3-(ter t-bu tyl)-3,4-d i-
h yd r o-2H-1,4-ben zoxa zin -5-yl] (p h en yl)m eth a n on e 5a. A
solution of compound 1 (244 mg, 1 mmol), TEAP (2.3 g, 10
mmol), and S-tert-leucinol (597 mg, 5 mmol) in methanol (500
mL) was oxidized, under nitrogen, at room temperature, at a
platinum electrode (E ) + 0.4 V vs sce). After exhaustive
oxidation, i.e., when a steady-state minimum value of the
current was recorded, benzylamine (5.3 g, 50 mmol) was added
to the oxidized solution and allowed to react for 30 min. Then,
the platinum anode was replaced by a mercury pool, and the

Synthesis of 8-Amino-1,4-benzoxazine Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5049
resulting solution was reduced at - 1.0 V vs sce. After
exhaustive cathodic electrolysis, the solution was poured into
a molar acetic acid buffered aqueous solution of pH ∼ 4.5 (100
mL). The resulting aqueous alcoholic solution was treated the
same as above (method A) and afforded, after chromatography
on silica with toluene as the eluent, compound 5a (120 mg,
30%) as an amorphous yellow solid that was recrystallized
from petroleum ether: mp 87-89 °C: 1H NMR [300 MHz,
(CD3)2SO] δ 1.00 (s, 9H, Me, But), 3.25 (m, 1H, 3-H), 3.85 (m,
1H, 2-CH2), 4.30 (m, 1H, 2-CH2), 4.40 (d, 2H, J ) 6 Hz, CH2,
benzyl), 5.85 (d, 1H, J ) 9 Hz, 6-H), 6.45 (t, 1H, J ) 6 Hz,
8-NH, D2O exchanged), 6.75 (d, 1H, J ) 9 Hz, 7-H), 7.10 to
7.30 (m, 5H, aromatic, benzyl), 7.45 (m, 5H, aromatic, 5-ben-
zoyl), 8.75 (s, 1H, 4-NH, D2O exchanged); 13C NMR [75 MHz,
(CD3)2SO] δ 26.9 (Me, But), 34.0 (CQ, But), 46.4 (CH2, benzyl),
58.3 (C-3), 65.9 (C-2), 100.1 (C-7), 109.1 (C-5), 127.6 (C-8a),
127.8 to 130.9 (CH, aromatic, benzyl and 5-benzoyl), 139.8 (CQ,
5-benzoyl), 141.2 (CQ, benzyl), 142.0 and 142.3 (C-4a and C-8),
197.0 (CO, methanone); MS (DCI) m/z ) 401 (MH+). Anal.
(C26H28N2O2) C, H, N.
(100 mL). The resulting hydroalcoholic solution was treated
according to method A and afforded, after chromatography on
silica with toluene as the eluent, compound 5e (141 mg, 50%)
as an amorphous yellow solid that was recrystallized from
petroleum ether: mp 99-101 °C: 1H NMR [300 MHz, (CD3)2-
SO] δ 0.90 (d, 6H, J ) 6 Hz, Me, isopentyl), 1.25 (s, 6H, 3-Me),
1.45 (q, 2H, J ) 6 Hz, CH2, isopentyl), 1.60 (m, 1H, J ) 6 Hz,
CH, isopentyl), 3.15 (q, 2H, J ) 6 Hz, CH2N, isopentyl), 3.85
(s, 2H, 2-CH2), 5.75 (t, 1H, J ) 6 Hz, 8-NH, D2O exchanged),
5.95 (d, 1H, J ) 9 Hz, 7-H), 6.85 (d, 1H, J ) 9 Hz, 6-H), 7.45
(m, 5H, aromatic, Ph), 8.40 (s, 1H, 4-NH, D2O exchanged); 13
C
NMR [75 MHz, (CD3)2SO] δ 23.5 (Me, isopentyl), 26.4 (CH,
isopentyl), 26.5 (3-Me), 39.1 (CH2, isopentyl), 48.6 (C-3 and
CH2N, isopentyl), 73.5 (2-CH2), 99.4 (C-7), 108.8 (C-5), 126.5
(C-8a), 129.0 (CH, meta, Ph), 129.1 (CH, ortho, Ph), 130.8 and
131.1 (C-6 and CH, para, Ph), 138.2 (CQ, Ph), 142.2 and 142.6
(C-4a and C-8), 196.4 (CO, methanone); MS (DCI) m/z ) 353
(MH+). Anal. (C22H28N2O2) C, H, N.
Meth od E. (3,3-Dim eth yl-8-h yd r oxyeth yla m in o-3,4-d i-
h yd r o-2H-1,4-ben zoxa zin -5-yl)(p h en yl)m eth a n on e 5f. A
solution of (R,S)-5-benzoyl-3,3-dimethyl-8a-hydroxy-3,4,8,8a-
tetrahydro-2H-1,4-benzoxazin-8-one (225 mg, 0.75 mmol) and
ethanolamine (2.5 mL, 37.5 mmol) in methanol (400 mL) was
allowed to react for 2h. Then, 7 mL of concentrated acetic acid
as well as zinc dust (250 mg, 3.7 mmol) were added, and the
resulting mixture was stirred for 5 min at room temperature.
The reaction mixture was filtered, and water (100 mL) was
then added. The resulting aqueous alcoholic solution was
treated the same as above (method A) and led to compound 5f
(147 mg, 60%) as an amorphous yellow solid that was crystal-
lized from pentane: mp 134-136 °C: 1H NMR [300 MHz,
(CD3)2SO] δ 1.25 (s, 6H, Me), 3.15 (q, 2H, J ) 6 Hz, CH2N,
hydroxyethyl), 3.55 (q, 2H, J ) 6 Hz, CH2N, hydroxyethyl),
3.85 (s, 2H, 2-CH2), 4.80 (t, 1H, J ) 6 Hz, OH, D2O exchanged),
5.65 (t, 1H, J ) 6 Hz, 8-NH, D2O exchanged), 6.00 (d, 1H, J )
9 Hz, 7-H), 6.85 (d, 1H, J ) 9 Hz, 6-H), 7.50 (m, 5H, aromatic,
Ph), 8.40 (s, 1H, 4-NH); 13C NMR [75 MHz, (CD3)2SO] δ 26.5
(Me), 45.6 (CH2N), 48.7 (C-3), 60.7 (CH2O), 73.5 (C-2), 99.6
(C-7), 109.0 (C-5), 126.6 (C-8a), 129.0, 129.1, 130.9 and 131.0
(C-6 and CH, aromatic, Ph), 138.2 (CQ, Ph), 142.1 and 142.6
(C-4a and C-8), 196.5 (CO, methanone); MS (DCI) m/z ) 327
(MH+). Anal. (C19H22N2O3) C, H, N.
{3,3-Dim eth yl-8-[2-h yd r oxy-1-(h yd r oxym eth yl)eth yl]-
am in o-3,4-dih ydr o-2H-1,4-ben zoxazin -5-yl}(ph en yl)m eth -
a n on e 5g. Compound 5g was prepared following a procedure
analogous to method D (see Table 2): 1H NMR [300 MHz,
(CD3)2SO] δ 1.25 (s, 6H, Me), 3.35 (m, 5H, CH2OH and CH,
8-amino chain), 3.85 (s, 2H, 2-CH2), 4.80 (s, 2H, OH, D2O
exchanged), 5.30 (d, 1H, J ) 6 Hz, 8-NH, D2O exchanged), 6.05
(d, 1H, J ) 9 Hz, 7-H), 6.85 (d, 1H, J ) 9 Hz, 6-H), 7.50 (m,
5H, aromatic, Ph), 8.40 (s, 1H, 4-NH, D2O exchanged); 13C
NMR [75 MHz, (CD3)2SO] δ 26.5 (Me), 48.7 (C-3), 56.0 (CH,
8-amino chain), 61.0 (CH2OH, 8-amino chain), 73.6 (C-2), 99.8
(C-7), 109.0 (C-5), 126.6 (C-8a), 129.0, 129.1 and 130.9 (C-6
and CH, aromatic Ph), 138.4 (CQ, Ph), 141.9 and 142.1 (C-4a
and C-8), 197.0 (CO, methanone); MS (DCI) m/z ) 357 (MH+).
Anal. (C20H24N2O4) C, H, N.
Compounds 5b-d were prepared following a procedure
analogous to method C (see Table 2).
(R,S)-(3-ter t-Bu t yl-8-p h en ylet h yla m in o-3,4-d ih yd r o-
1
2H-1,4-ben zoxa zin -5-yl)(p h en yl)m eth a n on e 5b: H NMR
[300 MHz, (CD3)2SO] δ 1.00 (s, 9H, Me, But), 2.85 (t, 2H, J )
8 Hz, CH2Ph, phenylethyl), 3.25 (m, 1H, 3-H), 3.35 (q, 2H, J
) 8 Hz, CH2N, phenylethyl), 3.90 (m, 1H, 2-CH2), 4.30 (dd,
1H, J ) 3 Hz, J ) 11 Hz, 2-CH2), 5.75 (t, 1H, J ) 6 Hz, 8-NH,
D2O exchanged), 6.05 (d, 1H, J ) 9 Hz, 7-H), 6.90 (d, 1H, J )
9 Hz, 6-H), 7.25 (m, 5H, aromatic, phenylethyl), 7.50 (m, 5H,
aromatic, 5-benzoyl), 8.80 (s, 1H, 4-NH, D2O exchanged); 13C
NMR [75 MHz, (CD3)2SO] δ 26.90 (Me, But), 34.05 (CQ, But),
36.40 (CH2Ph, phenylethyl), 44.80 (CH2N, phenylethyl), 58.20
(C-3), 65.80 (C-2), 99.70 (C-7), 109.5 (C-5), 127.2 to 130.9 (C-6
and CH, aromatic, phenylethyl and 5-benzoyl), 127.5 (C-8a),
139.8 (CQ, 5-benzoyl), 140.2 (CQ, phenylethyl), 142.0 and 142.2
(C-4a and C-8), 197.0 (CO, methanone); MS (DCI) m/z ) 415
(MH+). Anal. (C27H30N2O2) C, H, N.
(R,S)-(3-ter t-Bu t yl-8-isop en t yla m in o-3,4-d ih yd r o-2H -
1,4-ben zoxa zin -5-yl)(p h en yl)m eth a n on e 5c: 1H NMR [300
MHz, (CD3)2SO] δ 0.90 (d, 6H, J ) 6 Hz, Me, isopentyl), 1.00
(s, 9H, Me, But), 1.40 (q, 2H, J ) 8 Hz, CH2, isopentyl), 1.60
(m, 1H, J ) 6 Hz, CH, isopentyl), 3.10 (q, 2H, J ) 8 Hz, CH2N,
isopentyl), 3.25 (m, 1H, 3-H), 3.90 (m, 1H, 2-CH2), 4.25 (dd,
1H, J ) 4 Hz, J ) 11 Hz, 2-CH2), 5.65 (t, 1H, J ) 6 Hz, 8-NH,
D2O exchanged), 5.95 (d, 1H, J ) 9 Hz, 7-H), 6.85 (d, 1H, J )
9 Hz, 6-H), 7.50 (m, 5H, aromatic, Ph), 8.75 (s, 1H, 4-NH, D2O
exchanged); 13C NMR [75 MHz, (CD3)2SO] δ 23.50 (Me,
isopentyl), 26.40 (CH, isopentyl), 26.9 (Me, But), 34.0 (CQ, But),
39.1 (CH2, isopentyl), 41.2 (CH2N, isopentyl), 58.3 (C-3), 65.8
(2-CH2), 99.5 (C-7), 108.8 (C-5), 127.4 (C-8a), 129.0, 129.2 and
130.9 (C-6 and CH, aromatic, Ph), 139.7 (CQ, Ph), 142.1 and
142.5 (C-4a and C-8), 196.7 (CO, methanone); MS (DCI) m/z
) 381 (MH+). Anal. (C24H32N2O2) C, H, N.
(8-Ben zyla m in o-3,3-d im eth yl-3,4-d ih yd r o-2H-1,4-ben z-
oxa zin -5-yl)(p h en yl)m eth a n on e 5d : 1H NMR [300 MHz,
(CD3)2SO] δ 1.30 (s, 6H, 3-Me), 3.90 (s, 2H, 2-CH2), 4.40 (d,
2H, J ) 6 Hz, CH2, benzyl), 5.85 (d, 1H, J ) 9 Hz, 7-H), 6.55
(t, 1H, J ) 6 Hz, 8-NH, D2O exchanged), 6.75 (d, 1H, J ) 9
Hz, 6-H), 7.10 to 7.30 (m, 5H, aromatic, benzyl), 7.35 to 7.50
(m, 5H, aromatic, 5-benzoyl), 8.40 (s, 1H, 4-NH, D2O ex-
changed); MS (DCI) m/z ) 373 (MH+). Anal. (C24H24N2O2) C,
H, N.
Meth od D. (3,3-Dim eth yl-8-isop en tyla m in o-3,4-d ih y-
d r o-2H-1,4-ben zoxa zin -5-yl)(p h en yl)m eth a n on e 5e. A so-
lution of (R,S)-5-benzoyl-3,3-dimethyl-8a-hydroxy-3,4,8,8a-
tetrahydro-2H-1,4-benzoxazin-8-one (235 mg, 0.8 mmol), TEAP
(1.8 g, 8 mmol), and isopentylamine (4.5 mL, 40 mmol) in
methanol (400 mL) was allowed to react for 30 min. Then, the
resulting solution was reduced, under nitrogen, at room
temperature, at a mercury pool (E ) -1.0 V vs sce). After
exhaustive reduction, i.e., when a steady-state minimum value
of the current was recorded, the resulting solution was poured
into a molar acetic acid buffered aqueous solution of pH ∼ 4.5
Compounds 5h -n were prepared following a procedure
analogous to method C (see Table 2).
(8-Ben zyla m in o-3-sp ir o-1′-cyclop en tyl-3,4-d ih yd r o-2H-
1,4-ben zoxa zin -5-yl(p h en yl)m eth a n on e 5h . 1H NMR [300
MHz, (CD3)2SO] δ 1.60 to 1.80 (m, 8H, CH2, cyclopentyl), 4.00
(s, 2H, 2-CH2), 4.40 (d, 2H, CH2, benzyl), 5.90 (d, 1H, J ) 9
Hz, 7-H), 6.50 (t, 1H, J ) 6 Hz, 8-NH, D2O exchanged), 6.75
(d, 1H, J ) 9 Hz, 6-H), 7.10 to 7.25 (m, 5H, aromatic, benzyl),
7.40 (m, 5H, aromatic, 5-benzoyl), 8.55 (s, 1H, 4-NH, D2O
exchanged); 13C NMR [75 MHz, (CD3)2SO] δ 24.5 tï 37.7 (CH2,
cyclopentyl), 46.4 (CH2, benzyl), 59.8 (C-3), 71.6 (C-2), 100.1
(C-7), 109.2 (C-5), 127.3 (C-8a), 127.7, 128.0, 129.1, 129.4, 130.7
and 130.9 (C-6 and CH, aromatic, benzyl and 5-benzoyl), 138.6
(CQ, 5-benzoyl), 141.2 (CQ, 5-benzyl), 142.0 and 142.4 (C-4a
and C-8), 196.0 (CO, methanone); MS (DCI) m/z ) 399 (MH+).
Anal. (C26H26N2O2) C, H, N.
(8-Isob u t yla m in o-3-sp ir o-1′-cyclop en t yl-3,4-d ih yd r o-
2H-1,4-ben zoxa zin -5-yl)(p h en yl)m eth a n on e 5i: 1H NMR

5050 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Largeron et al.
[300 MHz, (CD3)2SO] δ 0.90 (d, 6H, Me, isobutyl), 1.50 tï 1.90
(m, 9H, CH, isobutyl and CH2, cyclopentyl), 2.95 (t, 2H, J ) 6
Hz, CH2, isobutyl), 3.95 (s, 2H, 2-CH2), 5.80 (t, 1H, J ) 6 Hz,
8-NH, D2O exchanged), 5.95 (d, 1H, J ) 9 Hz, 7-H), 6.85 (d,
1H, J ) 9 Hz, 6-H), 7.45 (m, 5H, aromatic, Ph), 8.55 (s, 1H,
4-NH, D2O exchanged); 13C NMR [75 MHz, (CD3)2SO] δ 21.2
(Me, isobutyl), 24.5 and 37.7 (CH2, cyclopentyl), 28.9 (CH,
isobutyl), 50.7 (CH2, isobutyl), 59.7 (C-3), 71.6 (2-CH2), 99.7
(C-7), 108.8 (C-5), 127.0 (C-8a), 129.0, 129.1 and 130.8 (C-6
and CH, aromatic Ph), 138.5 (CQ, Ph), 142.1 and 142.8 (C-4a
and C-8), 197.0 (CO, methanone); MS (DCI) m/z ) 365 (MH+).
Anal. (C23H28N2O2) C, H. N.
(8-Ben zyla m in o-3,3-d ip h en yl-3,4-d ih yd r o-2H-1,4-ben z-
oxa zin -5-yl)(p h en yl)m eth a n on e 5j: 1H NMR [300 MHz,
(CD3)2SO] δ 4.35 (d, 2H, J ) 6 Hz, CH2, benzyl), 4.80 (s, 2H,
2-CH2), 5.90 (d, 1H, J ) 9 Hz, 7-H), 6.55 (t, 1H, J ) 6 Hz,
8-NH, D2O exchanged), 6.85 (d, 1H, J ) 9 Hz, 6-H), 7.10 to
7.30 (m, 5H, aromatic, benzyl), 7.35 to 7.55 (m, 15H, aromatic,
3-Ph and 5-benzoyl), 9.50 (s, 1H, 4-NH, D2O exchanged); 13C
NMR [75 MHz, (CD3)2SO] δ 46.4 (CH2, benzyl), 60.3 (C-3), 71.6
(C-2), 100.6 (C-7), 109.5 (C-5), 127.6 (C-8a), 127.8 to 131.2 (C-6
and CH, aromatic, 3-Ph, benzyl and 5-benzoyl), 138.0 (CQ,
5-benzoyl), 141.0 (CQ, benzyl), 141.7 and 142.6 (C-4a and C-8),
144.3 (CQ, 3-Ph), 196.0 (CO, methanone); MS (DCI): m/z ) 497
(MH+). Anal. (C34H28N2O2) H, N; C: calcd, 82.85; found, 82.01.
(3,3-Dip h en yl-8-isop en t yla m in o-3,4-d ih yd r o-2H -1,4-
ben zoxa zin -5-yl)(p h en yl)m eth a n on e 5k : 1H NMR [300
MHz, (CD3)2SO] δ 0.85 (d, 6H, J ) 6 Hz, Me), 1.40 (q, 2H, J )
8 Hz, CH2, isopentyl), 1.60 (m, 1H, J ) 8 Hz, CH, isopentyl),
3.10 (q, 2H, J ) 8 Hz, CH2N, isopentyl), 4.70 (s, 2H, 2-CH2),
5.80 (t, 1H, J ) 6 Hz, 8-NH, D2O exchanged), 6.00 (d, 1H, J )
9 Hz, 7-H), 6.95 (d, 1H, J ) 9 Hz, 6-H), 7.20 to 7.40 (m, 10H,
aromatic, 3-Ph), 7.50 to 7.60 (m, 5H, aromatic, 5-benzoyl), 9.50
(s, 1H, 4-NH, D2O exchanged); 13C NMR [75 MHz, (CD3)2SO]
δ 23.5 (Me), 26.4 (CH, isopentyl), 38.9 (CH2, isopentyl), 41.2
(CH2N, isopentyl), 60.6 (C-3), 71.6 (C-2), 100.0 (C-7), 109.1 (C-
5), 127.3 (C-8a), 127.8 to 131.3 (C-6 and CH, aromatic, 3-Ph
and 5-benzoyl), 137.9 (CQ, 5-benzoyl), 141.8 and 142.8 (C-4a
and C-8), 144.4 (CQ, 3-Ph), 197.0 (CO, methanone); MS (DCI)
m/z ) 477 (MH+). Anal. (C32H32N2O2) C, H, N.
(R,S)-{8-[2-Hyd r oxy-1-(h yd r oxym eth yl)eth yl]a m in o-3-
h yd r oxym e t h yl-3,4-d ih yd r o-2H -1,4-b e n zoxa zin -5-yl}-
(p h en yl)m eth a n on e 5l: 1H NMR [300 MHz, (CD3)2SO] δ 3.40
to 3.60 (m, 8H, 3-CH2OH, CH2OH and CH, 8-amino chain),
4.10 (m, 2H, 2-CH2), 4.80 (t, 2H, J ) 6 Hz, OH, 8-amino chain,
D2O exchanged), 5.10 (t, 1H, J ) 6 Hz, 3-CH2OH, D2O
exchanged), 5.25 (d, 1H, J ) 8 Hz, 8-NH, D2O exchanged), 6.05
(d, 1H, J ) 9 Hz, 7-H), 6.85 (d, 1H, J ) 9 Hz, 6-H), 7.45 (m,
5H, aromatic, Ph), 8.60 (s, 1H, 4-NH, D2O exchanged); 13C
NMR [75 MHz, (CD3)2SO] δ 51.6 (C-3), 56.0 (CH, 8-amino
chain), 61.0 (CH2OH, 8-amino chain), 62.2 (3-CH2OH), 65.4
(C-2), 99.8 (C-7), 109.2 (C-5), 127.5 (C-8a), 129.0, 129.1 and
130.7 and 130.9 (C-6 and CH, aromatic, Ph), 138.4 (CQ, Ph),
141.9 and 142.1 (C-4a and C-8), 196.0 (CO, methanone); MS
(DCI) m/z ) 359 (MH+). Anal. (C19H22N2O5) C, H, N.
5-yl}(p h en yl)m eth a n on e 5n : 1H NMR [300 MHz, (CD3)2SO]
δ 1.20 (s, 3H, Me), 3.25 to 3.50 (m, 7H, 3-CH2OH, CH2OH and
CH, 8-amino chain), 3.70 (d, 1H, J ) 9 Hz, 2-CH2), 4.10 (d,
1H, J ) 9 Hz, 2-CH2), 4.80 (broad s, 2H, OH, 8-amino chain,
D2O exchanged), 5.10 (t, 1H, J ) 6 Hz, OH, 3-CH2OH, D2O
exchanged), 5.30 (d, 1H, J ) 6 Hz, 8-NH, D2O exchanged), 6.05
(d, 1H, J ) 9 Hz, 7-H), 6.85 (d, 1H, J ) 9 Hz, 6-H), 7.50 (m,
5H, aromatic, Ph), 8.50 (s, 1H, 4-NH, D2O exchanged); 13C
NMR [75 MHz, (CD3)2SO] δ 21.9 (Me), 52.7 (C-3), 56.0 (CH,
8-amino chain), 61.0 (CH2OH, 8-amino chain), 65.7 (C-2), 69.5
(3-CH2OH), 99.8 (C-7), 109.0 (C-5), 126.9 (C-8a), 129.0, 129.1
and 130.9 (C-6 and CH, aromatic, Ph), 138.5 (CQ, Ph), 141.9
and 142.1 (C-4a and C-8), 196.0 (CO, methanone); MS (DCI)
m/z ) 373 (MH+). Anal. (C20H24N2O5) C, H, N.
[8-(2-Hyd r oxyeth yla m in o-3,3-d ih yd r oxym eth yl-3,4-d i-
h yd r o-2H -1,4-b en zoxa zin -5-yl](p h en yl)m et h a n on e 5o:
Compound 5o was prepared following a procedure analogous
to method E (see Table 2): 1H NMR [300 MHz, (CD3)2SO] δ
3.15 (q, 2H, J ) 6 Hz, CH2N, hydroxyethyl), 3.35 (d, 2H, J )
6 Hz, 3-CH2OH), 3.50 (m, 4H, 3-CH2OH and CH2, hydroxy-
ethyl), 3.90 (s, 2H, 2-CH2), 4.80 (t, 1H, J ) 6 Hz, OH,
hydroxyethyl, D2O exchanged), 5.05 (t, 2H, J ) 6 Hz, OH,
3-CH2OH, D2O exchanged), 5.60 (t, 1H, J ) 6 Hz, 8-NH, D2O
exchanged), 5.90 (d, 1H, J ) 9 Hz, 7-H), 6.85 (d, 1H, J ) 9 Hz,
6-H), 7.40 (m, 5H, aromatic, Ph), 8.45 (s, 1H, 4-NH, D2O
exchanged); 13C NMR [75 MHz, (CD3)2SO] δ 45.6 (CH2N,
hydroxyethyl), 52.7 (C-3), 60.7 (CH2, hydroxyethyl), 65.7 (C-
2), 69.5 (3-CH2OH), 99.6 (C-7), 109.1 (C-5), 126.9 (C-8a), 129.0,
129.1, 130.9 and 131.0 (C-6 and CH, aromatic, Ph), 138.3 (CQ,
Ph), 142.1 and 142.5 (C-4a and C-8), 198.0 (CO, methanone);
MS (DCI) m/z ) 359 (MH+). Anal. (C19H22N2O5) C, H, N.
Meth od F . (8-Hyd r oxy-3-ter t-bu tyl-3,4-d ih yd r o-2H-1,4-
ben zoxa zin -5-yl)(p h en yl)m eth a n on e 6a . A solution of com-
pound 3a (327 mg, 1 mmol) and TEAP (2.3 g, 10 mmol) in
methanol (500 mL) was reduced, under nitrogen, at room
temperature, at a mercury electrode (E ) -1.3 V vs sce). After
exhaustive electrolysis, i.e., when a steady-state minimum
value of the current was recorded, the resulting solution was
evaporated to dryness, under reduced pressure, at 35 °C. The
residue was poured into water (50 mL) and extracted with
ethyl acetate (100 mL). The organic phase was dried (MgSO4)
and evaporated. The residue was chromatographed on silica
with toluene as the eluent, leading to compound 6a (265 mg,
85%) as an amorphous yellow solid that was recrystallized
from ether-pentane: mp 160-162 °C: 1H NMR [300 MHz,
(CD3)2SO] δ 1.00 (s, 9H, Me, But), 3.25 (m, 1H, 3-H), 3.90 (m,
1H, 2-CH2), 4.25 (m, 1H, 2-CH2), 6.10 (d, 1H, J ) 9 Hz, 7-H),
6.85 (d, 1H, J ) 9 Hz, 6-H), 7.50 (m, 5H, aromatic, Ph), 8.90
(s, 1H, NH, D2O exchanged), 9.95 (s, 1H, OH, D2O exchanged);
13C NMR [75 MHz, (CD3)2SO] δ 26.9 (Me, But), 34.1 (CQ, But),
58.2 (C-3), 65.4 (C-2), 105.4 (C-7), 110.9 (C-5), 129.1, 129.3 and
131.3 (CH, Ph), 129.6 (C-6), 130.4 (C-8a), 141.5 (CQ, Ph), 142.3
(C-4a), 150.9 (C-8), 197.0 (CO, methanone); MS (DCI) m/z )
312 (MH+). Anal. (C19H21NO3) C, H, N.
Meth od G. To a solution of compound 3a (327 mg, 1 mmol)
in 250 mL of methanol was added, at room temperature, 10
mL of concentrated acetic acid. Then, zinc dust (327 mg, 5
mmol) was added, and the resulting mixture was stirred for 2
min at room temperature. The reaction mixture was filtered,
and water (50 mL) was then added. The resulting solution was
concentrated to 50 mL under reduced pressure at 35 °C,
treated according to method F, and afforded compound 6a (186
mg, 60%).
(R,S)-[3-H yd r oxym et h yl-3-m et h yl-8-(2-h yd r oxyet h yl-
am in o)-3,4-dih ydr o-2H-1,4-ben zoxazin -5-yl](ph en yl)m eth -
a n on e 5m : 1H NMR [300 MHz, (CD3)2SO] δ 1.20 (s, 3H, Me),
3.15 (q, 2H, J ) 6 Hz, CH2N, hydroxyethyl), 3.25 (m, 1H,
3-CH2OH), 3.40 (m, 1H, 3-CH2OH), 3.50 (q, 2H, J ) 6 Hz, CH2,
hydroxyethyl), 3.70 (d, 1H, J ) 9 Hz, 2-CH2), 4.10 (d, 1H, J )
9 Hz, 2-CH2), 4.80 (t, 1H, J ) 6 Hz, OH, hydroxyethyl, D2O
exchanged), 5.15 (t, 1H, J ) 6 Hz, OH, 3-CH2OH, D2O
exchanged), 5.65 (t, 1H, J ) 6 Hz, 8-NH, D2O exchanged), 5.95
(d, 1H, J ) 9 Hz, 7-H), 6.80 (d, 1H, J ) 9 Hz, 6-H), 7.40 (m,
5H, aromatic, Ph), 8.45 (s, 1H, 4-NH, D2O exchanged); 13C
NMR [75 MHz, (CD3)2SO] δ 21.9 (Me), 45.6 (CH2N, hydroxy-
ethyl), 52.7 (C-3), 60.7 (CH2, hydroxyethyl), 65.7 (C-2), 69.5
(3-CH2OH), 99.6 (C-7), 109.1 (C-5), 126.9 (C-8a), 129.0, 129.1,
130.9 and 131.0 (C-6 and CH, aromatic, Ph), 138.3 (CQ, Ph),
142.1 and 142.5 (C-4a and C-8), 198.0 (CO, methanone); MS
(DCI) m/z ) 343 (MH+). Anal. (C19H22N2O4) C, H, N.
Compounds 6b and 6c were prepared following a procedure
analogous to method F or method G (see Table 1).
(8-Hyd r oxy-3-sp ir o-1′-cyclop en tyl-3,4-d ih yd r o-2H-1,4-
ben zoxa zin -5-yl)(p h en yl)m eth a n on e 6b: 1H NMR [300
MHz, (CD3)2SO] δ 1.50 to 1.80 (m, 8H, CH2, cyclopentyl), 3.90
(s, 2H, 2-CH2), 6.10 (d, 1H, J ) 9 Hz, 7-H), 6.85 (d, 1H, J ) 9
Hz, 6-H), 7.50 (m, 5H, aromatic, Ph), 8.70 (s, 1H, OH, D2O
exchanged), 9.95 (s, 1H, NH, D2O exchanged); 13C NMR [75
MHz, (CD3)2SO] δ 24.5 and 37.7 (CH2, cyclopentyl), 59.8 (C-
3), 71.3 (C-2), 105.4 (C-7), 111.1 (C-5), 129.1, 129.3 and 131.3
(R,S)-{3-Hyd r oxym eth yl-3-m eth yl-8-[2-h yd r oxy-1-(h y-
dr oxym eth yl)eth yl]am in o-3,4-dih ydr o-2H-1,4-ben zoxazin -

Synthesis of 8-Amino-1,4-benzoxazine Derivatives
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 5051
(CH, aromatic, Ph), 129.6 (C-6), 130.1 (C-8a), 141.0 and 141.6
(CQ, Ph and C-4a), 150.9 (C-8), 197.0 (CO, methanone); MS
(DCI) m/z ) 310 (MH+). Anal. (C19H19NO3) C, H, N.
were taken immediately at 540 nm with an automatic plate
reader (SLC 340ATTC, Labinstruments).
Ack n ow led gm en t. The authors thank Doctor Pierre
Renard, from ADIR, and Doctor Pierre Lestage, from
Institut de Recherche Servier, for fruitful discussions
in the course of this research.
[(R,S)-8-Hyd r oxy-3-h yd r oxym eth yl-3,4-d ih yd r o-2H-1,4-
b en zoxa zin -5-yl](p h en yl)m et h a n on e 6c: 1H NMR [300
MHz, (CD3)2SO] δ 3.00 to 3.70 (m, 3H and 3-CH2OH), 4.10 (s,
2H, 2-CH2), 5.10 (m, 1H, OH, CH2OH, D2O exchanged), 6.10
(d, 1H, J ) 9 Hz, 7-H), 6.85 (d, 1H, J ) 9 Hz, 6-H), 7.50 (m,
5H, aromatic, Ph), 8.75 (s, 1H, NH, D2O exchanged), 9.90 (s,
1H, 8-OH, D2O exchanged); 13C NMR [75 MHz, (CD3)2SO] δ
51.6 (C-3), 62.1 (CH2OH), 65.0 (C-2), 105.3 (C-7), 111.3 (C-5),
129.1, 129.2 and 131.3 (CH, aromatic, Ph), 129.6 (C-6), 130.4
(C-8a), 141.2 and 141.7 (CQ, Ph and C-4a), 150.9 (C-8), 198.0
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