Stille coupling reactions in the synthesis of hypoxia-selective 3-alkyl-1,2,4-benzotriazine 1,4-dioxide anticancer agents

Article abstract of DOI:10.1021/jo060986g

The introduction of a 3-alkyl substituent is a key step in the synthesis of 1,2,4-benzotriazine 1,4-dioxide hypoxia-selective anticancer agents, such as SN29751. The Stille reaction of 3-chloro-1,2,4-benzotriazine 1-oxides (BTOs) 5 was inhibited by the presence of electron donating substituents on the benzo ring, thus limiting the range of compounds available for SAR studies. The use of 3-iodo-BTOs 8 did not provide a significant improvement in the yields of 3-ethyl-BTOs 6. Microwave-assisted Stille coupling of chlorides 5 gave dramatically improved yields, which were consistently superior to those from the corresponding iodides 8. The application of microwave-assisted synthesis extended the range of substituted BTOs available for SAR studies and provided an efficient, scalable synthesis of the investigational anticancer agent, SN29751 (1).

Full text of DOI:10.1021/jo060986g

Stille Coupling Reactions in the Synthesis of Hypoxia-Selective
3-Alkyl-1,2,4-Benzotriazine 1,4-Dioxide Anticancer Agents
Karin Pchalek and Michael P. Hay*
Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences,
The UniVersity of Auckland, Auckland, New Zealand
m.hay@auckland.ac.nz
ReceiVed May 11, 2006
The introduction of a 3-alkyl substituent is a key step in the synthesis of 1,2,4-benzotriazine 1,4-dioxide
hypoxia-selective anticancer agents, such as SN29751. The Stille reaction of 3-chloro-1,2,4-benzotriazine
1-oxides (BTOs) 5 was inhibited by the presence of electron donating substituents on the benzo ring,
thus limiting the range of compounds available for SAR studies. The use of 3-iodo-BTOs 8 did not
provide a significant improvement in the yields of 3-ethyl-BTOs 6. Microwave-assisted Stille coupling
of chlorides 5 gave dramatically improved yields, which were consistently superior to those from the
corresponding iodides 8. The application of microwave-assisted synthesis extended the range of substituted
BTOs available for SAR studies and provided an efficient, scalable synthesis of the investigational
anticancer agent, SN29751 (1).
Introduction
As part of a program to develop bioreductive hypoxia-
selective anticancer agents1,2 based on the 1,2,4-benzotriazine
1,4-dioxide template we recently described3,4 a series of 3-alkyl
1,2,4-benzotriazine 1,4-dioxide derivatives with in vivo antitu-
mor activity in combination with radiation. One of these
analogues, SN29751 (1), in combination with radiation, has
superior in vivo activity compared to tirapazamine (2), which
is currently in Phase III clinical trial in combination with
chemoradiation.5-7
A key step in the synthesis of 1 and related compounds is the
introduction of the 3-alkyl substituent. Previous workers have
used the Bamberger synthesis8 to install 3-alkyl substituents as
part of the 1,2,4-benzotriazine ring formation. This unwieldy
approach, requiring the synthesis of the appropriately substituted
formazan, cyclization to the 1,2,4-benzotriazine and N-oxidation,
gave low9 or unreported10 yields of 3-alkyl-1,2,4-benzotriazine
1-oxides (BTOs). In a recent letter,11 we reported a more
(1) Brown, J. M.; Wilson, W. R. Nat. ReV. Cancer 2004, 4, 437-447.
(2) Hay, M. P.; Gamage, S. A.; Kovacs, M.; Pruijn, F. B.; Anderson, R.
F.; Patterson, A. V.; Wilson, W. R.; Brown, J. M.; Denny, W. A. J. Med.
Chem. 2003, 46, 169-182.
(3) Hay, M. P.; Pruijn, F. B.; Hicks, K. O.; Pchalek, K.; Botting, J.;
Hogg, A.; Petrovic-Stojanovska, B.; Liyange, H. D. S.; Valentine, S. P.;
Siim, B. G.; Denny, W. A.; Wilson, W. R. Abstract 3790, Proceedings of
the American Association for Cancer Research Annual Meeting: Anaheim,
CA, 2005.
(4) Siim, B. G.; Hay, M. P.; Pruijn, F. B.; Hicks, K. O.; Pchalek, K.;
Denny, W. A.; Valentine, S. P.; Fraser, A. M.; Wilson, W. R. Abstract
A32, Proceedings of AACR-NCI;EORTC International Conference on
Molecular Targets and Cancer Therapeutics: Discovery, Biology, and
Clinical Applications: Philadelphia, PA, 2005.
(5) Peters, L. J.; Rischin, D.; Hicks, R. J.; Hughes, P. G.; Sizeland, A.
M. Int. J. Radiat. Oncol. Biol. Phys. 1999, 45, 148-149.
(6) Rishin, D.; Peters, L.; Hicks, R.; Hughes, P.; Fisher, R.; Hart, R.;
Sexton, M.; D’Costa, I.; von Roemeling, R. J. Clin. Oncol. 2001, 19, 535-
542.
(7) Rischin, D.; Peters, L.; Fisher, R.; Macann, A.; Denham, J.; Poulsen,
M.; Jackson, M.; Kenny, L.; Penniment, M.; Corry, J.; Lamb, D.; McClure,
B. J. Clin. Oncol. 2005, 23, 79-87.
(8) Abramovitch, R. A.; Schofield, K. J. Chem. Soc. 1955, 2326-2336.
(9) Atallah, R. H.; Nazer, M. Z. Tetrahedron 1982, 38, 1793-1796.
(10) Kelson, A. B.; McNamara, J. P.; Pandey, A.; Ryan, K. J.; Dorie,
M. J.; McAfee, P. A.; Menke, D. R.; Brown, J. M.; Tracy, M. Anti-Cancer
Drug Des. 1998, 13, 575-592.
(11) Hay, M. P.; Denny, W. A. Tetrahedron Lett. 2002, 43, 9569-9571.
10.1021/jo060986g CCC: $33.50 © 2006 American Chemical Society
Published on Web 07/25/2006
6530
J. Org. Chem. 2006, 71, 6530-6535

Stille Coupling Reactions of Anticancer Agents
SCHEME 1a
TABLE 1. Stille Coupling of 3-Cl-BTOs 5a-k under Thermal
and Microwave-Assisted Conditionsa
thermal reaction
microwave-assisted
of 5 (%)b
reaction of 5 (%)b
entry
R1
R2
5c
6
time (min)
5c
6
a
b
c
d
e
f
g
h
i
H
Me
H
H
H
Me
Me
0
56
75
54
38
34
70
75
76
0
86d
37d
14
40
60
60
40
40
40
40
60
20
20
40
0
0
0
0
0
0
0
0
0
0
0
78
84
78
75
82
54
74
67
86
88
86
Me
30
-CH2CH2CH2-
MeO
H
MeO
Me
F
12d
28d
16
H
a Reagents: (a) NH2CN, concd HCl; (b) aq NaOH; (c) NaNO2,
CF3CO2H; (d) POCl3, DMF; (e) tert-BuNO2, I2, CuI, THF; (f) Et4Sn,
Pd(PPh3)4, DME; (g) CF3CO3H, DCM.
MeO
Me
MeO
H
14
14
j
k
75d
60d
versatile approach using Stille coupling12 of a 3-chloro-BTO
5a with Et4Sn to give the corresponding 3-ethyl-BTO 6a (R1
) R2 ) H; Scheme 1) in good yield, which could then be
oxidized to the 1,4-dioxide 7.
H
F
0
a All reactions were carried out on a 0.4 mmol scale under N2 with the
same batch of catalyst. b Isolated yields. c Recovered starting material.
d Addition of extra portion of Pd(PPh3)4 after 6 h.
We wished to include alkyl and alkoxy moieties at the 6-
and 7-position of these 3-alkyl-BTOs in order to modulate the
reduction potentials and, consequently, the potency of the
compounds2 and also to link amine side chains in order to
increase aqueous solubility. In this report we describe our efforts
to extend the scope and utility of the Stille reaction in the 1,2,4-
benzotriazine 1-oxide system. We have applied the Stille
reaction to a variety of alkyl- and alkoxy-substituted BTOs,
which were required for SAR studies, and have explored the
application of microwave-assisted synthesis to this transforma-
tion. We also report the use of these results in the development
of an efficient, scalable synthesis of 1.
and 7-positions, incomplete reactions and low product yields
(14-37%) were observed, irrespective of further catalyst
addition (Table 1).
Our first approach to remedy this shortcoming was to consider
more active catalysts. The reaction of 5e and 5f with Pd2(dba)3/
PtBu3/CsF in dioxane18 or Pd(dba)3/P(2-furyl)3/LiCl in DME19
gave only traces of products 6e and 6f, respectively. The reaction
of 5e with Pd(dppf)Cl2/DME gave only poor yields of the
corresponding 6e. Activation of the 3-chloride 5a by oxidation
to the corresponding 1,4-dioxide was attempted, but the product
was insufficiently stable to isolate. Our next strategy was to
modify the halogen atom since it is known that oxidative
addition of Pd0 to the strong carbon-chloride bond is disfavored
because of the higher bond dissociation energy (Ph-Cl is 96
kcal mol-1 compared with 81 kcal mol-1 for Ph-Br and 65 kcal
mol-1 for PhI).20 Attempts to form the 3-bromo derivatives by
diazotization of the 3-amine 4e with NaNO2 in a mixture of
48% HBr and DMF gave a very low yield of the corresponding
bromide.21 In contrast, reaction of BTO-3-amines 4a-i and tert-
butyl nitrite with a mixture of I2 and CuI in THF at reflux
temperature gave better yields (52-82%) of the 3-iodides 8a-i
(Scheme 1).
However, reaction of iodides 8a-i under identical conditions
described for chlorides 5a-i gave mostly reduced yields or
modest increases (-42 to 17%) of 6a-i (Figure 1).22 Further-
more, the overall mass recovery of starting material and product
was consistently lower for the iodides 8 (49-83% mass
recovery) compared to the chlorides 5 (50-93% mass recovery).
This observation, as well as the presence of a variety of
additional, unidentified byproducts in the coupling reaction of
the iodides 8 indicated that the 3-iodo-BTOs 8 are unstable under
Results and Discussion
Stille Coupling of Chlorides 5a-k and Iodides 8a-k under
Conventional Heating. A variety of alkyl- and alkoxy-
substituted BTOs were required for SAR studies13 (Table 1),
and the use of the previously defined Stille methodology was
considered to be most appropriate to provide these compounds.
Thus, nitroanilines 3a-i were converted to BTO-3-amines 4a-i
using the Mason and Tennant modification14 of the method of
Arndt (Scheme 1).15 Subsequent diazotization, and chlorination
of the intermediate phenols, gave the 3-chlorides 5a-i in
moderate to good yields (49-95%). The Stille reaction of BTO
16
5a with Et4Sn using Pd(PPh3)4 as the catalyst in refluxing
DME for 20 h gave good yields (Table 1), although the reaction
stalled after 6 h and required the addition of another batch of
catalyst.17 However, with the introduction of electron donating
alkyl and methoxy substituents (compounds 5b-i) into the 6-
(12) (a) Stille, J. K. Angew. Chem., Int. Ed. Engl. 1986, 25, 508-524.
(b) Stille, J. K. Pure Appl. Chem. 1985, 57, 1771-1780.
(13) The results of these biological studies will be reported in a
forthcoming article.
(14) Mason, J. C.; Tennant, G. J. Chem. Soc. B 1970, 911-916.
(15) Arndt, F. Ber. Dtsch. Chem. Ges. 1914, 46, 3522-3530.
(16) During the course of the study we found considerable variation in
the efficacy of the Pd(PPh3)4 among different batches from suppliers. There
appeared to be no relationship between the activity of the catalyst and its
physical appearance. The data in this article were generated using the same
batch of catalyst.
(18) Littke, A. F.; Schwarz, L.; Fu, G. C. J. Am. Chem. Soc. 2002, 124,
6343-6348. Littke, A. F.; Fu, G. C. Angew. Chem., Int. Ed. Engl. 1999,
38, 2411-2413.
(19) Farina, V.; Krishnan, B. J. Am. Chem. Soc. 1991, 113, 9585-9595.
(20) Mee, S. H. P.; Lee, V.; Baldwin, J. E. Chem.sEur. J. 2005, 11,
3294-3308.
(21) Personal communication, Dr H. H. Lee, ACSRC, The University
of Auckland.
(17) Thermal Stille reactions were carried out in a 12 port carousel reactor
to ensure identical reaction conditions.
(22) Full tables with yields of starting material and product are available
in the Supporting Information.
J. Org. Chem, Vol. 71, No. 17, 2006 6531

Pchalek and Hay
TABLE 2. Optimization of Microwave-Assisted Stille Coupling of
5ja
T
time
solvent (°C) (min) 6j (%)b
comments
1
2
3
4
5
6
DME
DME
DME
DMF
MeCN
MeCN
130
140
150
140
130
140
60
20
10
20
60
40
47
78
50
<80
43
incomplete reaction
unstable temperature
reaction incomplete, byproducts
inseparable mixture
incomplete reaction
reproducible 0.1-1.0 g
85
a All reactions were carried out on a 0.5 mmol scale with the same batch
of catalyst. b Isolated yields.
TABLE 3. Catalyst Selection for Microwave-Assisted Stille
Coupling of 5ja
time
(min)
6j
catalystb
Pd/C
Pd(dppf)Cl2
Pd(CH3CN)2Cl2
Pd(OAc)2
(%)c
comments
no reaction
no reaction
no reaction
mainly unreacted sm
incomplete reaction
time varied with batch
1
2
3
4
5
6
40
40
40
60
60
40
0
0
0
trace
43
85
Pd(PPh3)2Cl2
Pd(PPh3)4
FIGURE 1. Stille coupling of chlorides 5a-k and iodides 8a-k.
SCHEME 2a
a All reactions were carried out in MeCN at 140 °C on a 0.5 mmol scale.
b Catalyst loading of 5 mol %. c Isolated yields.
ired byproducts, and long reaction times. Consequently, the use
of microwave conditions with the Stille reaction of the 3-halo-
BTOs and Et4Sn was investigated. The 3-chloro-6-fluoro-BTO
5j was used to optimize the reaction conditions (Table 2). Three
solvents, DME, DMF, and MeCN, covering a range of polarity,
were tested at 130, 140, and 150 °C. The highest yield of 6j in
DME was achieved at 140 °C, with lower temperatures giving
incomplete conversion, even under prolonged reaction times,
and higher temperatures showing significant formation of
byproducts even with shorter reaction times. However, difficulty
in maintaining the required temperature profile when using
DME, because of its relatively low dielectric constant, resulted
in poor reproducibility. The use of DMF as a solvent gave
byproducts which could not be separated from the product by
column chromatography. Reactions in MeCN needed longer
reaction times compared to those carried out in DME and DMF,
but MeCN proved to be the solvent of choice, providing clean
and reproducible conversion to 6j in 85% yield.
The variation in yields and conversion rates seen with
different batches of Pd(PPh3)4 using thermal heating was less
pronounced under microwave conditions (Table 3, entry 6), with
some batches requiring longer reaction times but without
lowering the yield. Nevertheless, this variation prompted us to
examine several other catalysts with the aim to improve the
reproducibility of the reaction. Again, 3-chloro-6-fluoro-BTO
5j was used with a variety of Pd catalysts (Table 3). The use of
Pd/C, Pd(dppf)Cl2 and Pd(CH3CN)2Cl2 catalysts did not give
any conversion at all; Pd(OAc)2 showed only a trace of 6j;
whereas Pd(PPh3)2Cl2 gave incomplete conversion to yield 43%
of 6j, which was not improved by the addition of LiCl to the
reaction.
a Reagents: (a) NaNO2, CF3CO2H; (b) POCl3, DMF; (c) tert-BuNO2,
I2, CuI, THF; (d) Et4Sn, Pd(PPh3)4, DME; (e) NaOMe, MeOH.
the coupling conditions and undergo substantial decomposition.
An alternative approach to access the methoxy derivatives 6f
and 6g was considered (Scheme 2). The 6-fluoro-3-amine 4j
and the 7-isomer 4k were converted to the 3-chlorides 5j and
5k, respectively, and also the corresponding 3-iodides 8j and
8k. The chlorides 5j and 5k underwent Stille coupling to give
6j and 6k in 75% and 60% yields, respectively (Table 1). In
contrast, iodides 8j and 8k gave incomplete conversion and low
yields (29 and 27%, respectively) of the corresponding com-
pounds.22 Displacement of the fluorides 6j and 6k with
methoxide gave 3-ethyl-BTOs 6f and 6g in excellent yield, 95
and 91%, respectively (Scheme 2).
Microwave-Assisted Stille Coupling of Chlorides and
Iodides. The use of microwave-assisted synthesis23 with ho-
mogeneous catalysis has been well documented and has been
used to overcome the disadvantages of thermal heating, such
as low yields for electron-rich derivatives, formation of undes-
The complete set of 3-chloro-BTOs 5 and 3-iodo-BTOs 8
were processed using Pd(PPh3)4 in MeCN at 140 °C. Reaction
of the chlorides 5 proceeded smoothly to completion within 20-
60 min, giving yields between 74 and 88%, with the exception
of the 6-OMe substituted chlorides 5f and 5h where the yields
were 54% and 67%, respectively (Table 1). The iodides 8
showed a similar pattern, but lower overall yields (57-73%)
(23) (a) Olofsson, K.; Larhed, M. In MicrowaVe Assisted Organic
Synthesis; Tierney, J. P., Lidsrom, P., Eds.; Blackwell Publishing: Oxford,
U.K., 2005; Chapter 2, p 23-43. (b) Kappe, C. O. Angew. Chem., Int. Ed.
2004, 43, 6250-6284. (c) Larhed, M.; Moberg, C.; Hallberg, A. Acc. Chem.
Res. 2002, 35, 717-727 and references therein.
6532 J. Org. Chem., Vol. 71, No. 17, 2006

Stille Coupling Reactions of Anticancer Agents
SCHEME 3a
until the mixture was strongly basic, and the mixture was stirred at
100 °C for 3 h. The mixture was cooled, diluted with water (100
mL), filtered, washed with water (3 × 30 mL), washed with ether
(2 × 5 mL), and dried. If necessary, the product was purified by
chromatography, eluting with a gradient (0-10%) of MeOH/DCM,
to give amine 4. Compounds 4a-c, 4f, 4g, 4j, and 4k were prepared
as previously described.2
6,7-Dimethyl-1,2,4-benzotriazin-3-amine 1-Oxide (4d). Reac-
tion of 4,5-dimethyl-2-nitroaniline (3d) gave amine 4d (97%) as a
1
yellow solid: mp 284-286 °C (lit.14 mp 286 °C dec). H NMR:
δ 7.91 (s, 1H), 7.34 (s, 1H), 7.15 (br s, 2H), 2.36 (s, 3H), 2.33 (s,
3H).
Preparation of Chlorides (5a-k). General Method. Sodium
nitrite (22 mmol) was added in small portions to a stirred solution
of 1-oxide 4 (20 mmol) in TFA (40 mL) at 5 °C, and the solution
stirred at 20 °C for 3 h. The solution was poured into ice/water
(400 mL), stirred 30 min, filtered, washed with water (3 × 10 mL),
and dried. The solid was suspended in POCl3 (50 mL) and DMF
(0.25 mL) and stirred at 100 °C for 1 h. The solution was cooled,
poured into ice/water (500 mL), stirred for 30 min, filtered, washed
with water (3 × 20 mL) and dried. The filtrate was extracted with
EtOAc (3 × 70 mL) and dried, and the solvent was evaporated.
The combined residues were purified by chromatography, eluting
with 5% EtOAc/DCM to give chloride 5.
a Reagents: (a) Et4Sn, Pd(PPh3)4, DME; (b) MORPHCH2CH2CH2OH,
NaH, THF; (c) CF3CO3H, CF3CO2H, DCM.
were observed, with the exception of 8a and the 6-MeO
substituted iodides 8f and 8h where complete conversion could
not be achieved after 60 min and low yields (33, 11, and 17%,
respectively) were observed (Figure 1). The use of microwave-
assisted reaction conditions allows Stille reactions to be carried
out in good yields even on relatively electron-rich BTO chlorides
5, allowing shorter reaction times, higher product yields, and
cleaner reactions. Interestingly, the use of 3-iodides 8 in the
Stille reaction did not provide any clear benefit in conjunction
with either thermal or microwave heating, with the reactivity
of the iodide under the reaction conditions leading to byproducts.
Evaluation of the data produced from the reaction array
suggested that the most efficient approach to 6-alkoxy substi-
tuted BTO derivatives such as 1 would be via microwave-
assisted Stille coupling of chloride 5j followed by displacement
of the fluoride 6j with an alkoxide (Scheme 3). Thus, reaction
of 5j with Et4Sn on a 9 × 1 g scale under microwave heating
gave a combined yield of 84%. Alkylation of 6j with the side
chain alkoxide gave 1-oxide 9 in 76% yield. Selective aromatic
N-oxidation at the 4-position was achieved in modest yield using
an excess of trifluoroperacetic acid in the presence of trifluor-
acetic acid, which acts to protect the morpholine side chain from
oxidation.
3-Chloro-1,2,4-benzotriazine 1-Oxide (5a). Reaction of amine
4a gave chloride 5a (95%) as a pale yellow powder: mp (DCM)
1
119-119.5 °C; [lit.24 (MeOH) 117-118 °C]. H NMR [(CD3)2-
SO]: δ 8.38 (dd, J ) 8.7, 1.0 Hz, 1H), 8.16 (ddd, J ) 8.3, 7.0, 1.3
Hz, 1H), 8.06 (dd, J ) 8.2, 1.0 Hz, 1H), 7.90 (ddd, J ) 8.7, 6.9,
1.3 Hz, 1H). 13C NMR [(CD3)2SO]: δ 155.3 (C), 146.9 (C), 137.2
(CH), 133.9 (C), 131.5 (CH), 128.0 (CH), 119.9 (CH).
Stille Coupling of Chlorides 5a-k. General Method. Pd(PPh3)4
(0.025 mmol) was added to a N2-purged, stirred solution of chloride
5 (0.50 mmol) and Et4Sn (0.60 mmol) in DME (10 mL). The
solution was degassed and stirred under N2 at reflux temperature
for 20 h. A second batch of Pd(PPh3)4 was added after 6 h if the
reaction appeared to have stopped by TLC. The solvent was
evaporated, and the residue was dissolved in DCM (10 mL) and
stirred with saturated aqueous KF solution (10 mL) for 30 min.
The mixture was filtered through Celite. The Celite was washed
with DCM, and the combined organic filtrate was washed with
water. The organic fraction was dried, and the solvent evaporated.
The residue was purified by chromatography, eluting with DCM
to give product, which was, if necessary, further purified by
chromatography, eluting with 20% EtOAc/petroleum ether, to give
3-ethyl-1-oxide 6.
Although the 3-chloro-BTO system 5 is activated toward
palladium-mediated coupling reactions, the presence of electron
donating substituents on the benzo ring is sufficiently deactivat-
ing to limit their synthetic utility in the Stille reaction. The use
of 3-iodo-BTOs 8 did not provide a significant improvement
in yield of 3-ethyl-BTOs 6. The use of microwave-assisted
synthesis in the Stille coupling of chlorides 5 gave dramatically
improved yields, which were consistently superior to those
obtained with the iodides 8. The application of microwave-
assisted synthesis extends the utility and scope of palladium-
mediated coupling reactions in this system and provides a more
efficient synthesis of the investigational anticancer agent,
SN29751 (1).
3-Ethyl-1,2,4-benzotriazine 1-Oxide (6a). Reaction of chloride
5a gave 1-oxide 6a (86%) as a white solid: mp (EtOAc/petroleum
ether) 78-80 °C. 1H NMR: δ 8.45 (dd, J ) 8.7, 1.1 Hz, 1H), 7.99
(dd, J ) 8.5, 1.1 Hz, 1H), 7.93 (ddd, J ) 8.5, 7.1, 1.3 Hz, 1H),
7.69 (ddd, J ) 8.7, 7.1, 1.2 Hz, 1H), 3.06 (q, J ) 7.6 Hz, 2H),
1.45 (t, J ) 7.6 Hz, 3H). 13C NMR: δ 168.1 (C), 147.6 (C), 135.5
(C), 133.2 (C), 129.8 (CH), 128.7 (CH), 120.1 (CH), 30.7 (CH2),
12.2 (CH3). Anal. Calcd for C9H9N3O3: C, 61.70; H, 5.18; N, 23.99.
Found: C, 61.33; H, 5.15; N, 23.72%.
3-Ethyl-6-methyl-1,2,4-benzotriazine 1-Oxide (6b). Reaction
of chloride 5b gave (i) starting material 5b (56%) and (ii) 1-oxide
6b (37%) as a white solid: mp (EtOAc/DCM) 68-70 °C. 1H
NMR: δ 8.33 (d, J ) 8.8 Hz, 1H), 7.74 (br s, 1H), 7.49 (dd, J )
8.8, 1.7 Hz, 1H), 3.02 (q, J ) 7.6 Hz, 2H), 2.59 (s, 3H), 1.44 (t, J
) 7.6 Hz, 3H). 13C NMR: δ 168.2 (C), 147.8 (C), 147.1 (C), 132.0
(CH), 131.6 (C), 127.5 (CH), 119.8 (CH), 30.7 (CH2), 22.1 (CH3),
12.2 (CH3). Anal. Calcd for C10H11N3O: C, 63.48; H, 5.86; N,
22.21. Found: C, 63.46; H, 6.03; N, 22.31%.
Experimental Section
Preparation of 3-Amino-1,2,4-benzotriazine 1-Oxides 4. Gen-
eral Method. A stirred mixture of nitroaniline 3 (20 mmol) and
NH2CN (80 mmol) were melted together at 100 °C, cooled to 50
°C. Concentrated HCl (10 mL) was added dropwise (CAUTION:
exotherm), and the mixture was heated at 100 °C for 4 h. The
mixture was cooled to 50 °C. A 7.5 M NaOH solution was added
3-Ethyl-7-methyl-1,2,4-benzotriazine 1-Oxide (6c). Reaction
of chloride 5c gave (i) starting material 5c (75%) and (ii) 1-oxide
6c (14%) as an off-white solid: mp (EtOAc/DCM) 128-131 °C.
(24) Robbins, R. F.; Schofield, K. J. Chem. Soc. 1957, 3186-3194.
J. Org. Chem, Vol. 71, No. 17, 2006 6533

Pchalek and Hay
1H NMR: δ 8.24 (br s, 1H), 7.87 (d, J ) 8.6 Hz, 1H), 7.74 (dd, J
) 8.6, 2.9 Hz, 1H), 3.04 (q, J ) 7.6 Hz, 2H), 2.58 (s, 3H), 1.44 (t,
J ) 7.6 Hz, 3H). 13C NMR: δ 167.3 (C), 153.2 (C), 146.1 (C),
141.1 (C), 137.6 (CH), 128.3 (CH), 118.8 (CH), 30.6 (CH2), 21.8
(CH3), 12.3 (CH3). Anal. Calcd for C10H11N3O: C, 63.48; H, 5.86;
N, 22.21. Found: C, 63.18; H, 5.74; N, 21.90%.
) 22 Hz, CH), 30.7 (CH2), 12.2 (CH3). Anal. Calcd for C9H8-
FN3O: C, 55.96; H, 4.17; N, 21.75. Found: C, 56.01; H, 4.20; N,
21.82%.
3-Ethyl-7-fluoro-1,2,4-benzotriazine 1-Oxide (6k). Reaction of
chloride 5k gave 1-oxide 6k (60%) as a pale yellow solid: mp
1
(EtOAc/petroleum ether) 91-92 °C. H NMR: δ 8.10 (dd, J )
6,7-Dimethyl-3-ethyl-1,2,4-benzotriazine 1-Oxide (6d). Reac-
tion of chloride 5d gave (i) starting material 5d (54%) and (ii)
1-oxide 6d (30%) as a pale-yellow solid: mp (EtOAc/DCM) 61-
63 °C. 1H NMR: δ 8.20 (s, 1H), 7.72 (s, 1H), 3.02 (q, J ) 7.6 Hz,
2H), 2.49 (s, 3H), 2.48 (s, 3H), 1.43 (t, J ) 7.6 Hz, 3H). 13C
NMR: δ 167.4 (C), 147.1 (C), 146.6 (C), 141.0 (C), 131.5 (C),
127.7 (CH), 119.0 (CH), 30.7 (CH2), 20.6 (CH3), 20.3 (CH3), 12.3
(CH3). Anal. Calcd for C11H13N3O: C, 65.01; H, 6.45; N, 20.68.
Found: C, 65.23; H, 6.27; N, 20.51%.
Ethyl-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1-Oxide (6e).
Reaction of chloride 5e gave (i) starting material 5e (38%) and (ii)
1-oxide 6e (12%) as a pale yellow solid: mp (MeOH) 80-81 °C.
1H NMR: δ 8.26 (s, 1H), 7.26 (s, 1H), 3.11 (dt, J ) 7.2 Hz, 4H),
3.02 (q, J ) 7.6 Hz, 2H), 2.21 (p, J ) 7.2 Hz, 2H), 1.43 (t, J ) 7.6
Hz, 3H). 13C NMR: δ 167.0 (C), 154.6 (C), 148.7 (C), 147.6 (C),
132.3 (C), 122.7 (CH), 114.3 (CH), 33.2 (CH2), 32.8 (CH2), 30.6
(CH2), 25.8 (CH2), 12.4 (CH3). Anal. Calcd for C12H13N3O‚1/4CH3-
OH: C, 65.90; H, 6.32; N, 18.82. Found: C, 66.06; H, 6.13; N,
18.51%.
7.9, 2.8 Hz, 1H), 8.02 (q, J ) 5.1 Hz, 1H), 7.67-7.72 (m, 1H),
3.06 (q, J ) 7.6 Hz, 2H), 1.45 (t, J ) 7.6 Hz, 3H). 13C NMR: δ
167.7 (d, J ) 2 Hz, C), 163.4 (C), 160.8 (C), 144.8 (C), 131.3 (d,
J ) 9 Hz, CH), 125.8 (d, J ) 26 Hz, CH), 105.0 (d, J ) 27 Hz,
CH), 30.6 (CH2), 12.2 (CH3). Anal. Calcd for C9H8FN3O: C, 55.96;
H, 4.17; N, 21.75. Found: C, 56.04; H, 4.21; N, 22.06%.
Preparation of Iodides 8a-k. General Method. tert-BuONO
(18 mmol) was added to a stirred solution of amine 4 (6 mmol), I2
(6 mmol), and CuI (0.6 mmol) in THF (100 mL), and the mixture
was stirred at reflux temperature for 2 h. The mixture was cooled
to 20 °C, and the mixture was filtered through a short plug of
alumina and washed with THF (100 mL). The filtrate was
evaporated. The residue was suspended in DCM (100 mL), washed
with aqueous Na2S2O5 solution (10%, 2 × 50 mL), water (50 mL),
and brine (50 mL), and dried, and the solvent was evaporated. The
residue was purified by chromatography, eluting with 5% EtOAc/
DCM, to give iodide 8.
3-Iodo-1,2,4-benzotriazine 1-Oxide (8a). Reaction of amine 4a
gave iodide 8a (73%) as a pale yellow powder: mp (EtOAc/DCM)
207-210 °C. 1H NMR: δ 8.37 (br d, J ) 8.3 Hz, 1H), 7.93-8.00
(m, 2H), 7.77 (ddd, J ) 8.5, 6.3, 2.3 Hz, 1H). 13C NMR: δ 147.5
(C), 136.3 (CH), 134.4 (C), 131.1 (CH), 128.4 (CH), 123.1 (C),
120.3 (CH). Anal. Calcd for C7H4IN3O: C, 30.79; H, 1.48; N,
15.39. Found: C, 31.09; H, 1.39; N, 15.52%.
Stille Coupling of Iodides 8a-k. General Method. Pd(PPh3)4
(0.020 mmol) was added to a N2-purged, stirred solution of iodide
7 (0.40 mmol) and Et4Sn (0.24 mmol) in DME (10 mL). The
solution was degassed and stirred under N2 at reflux temperature
for 16 h. The solvent was evaporated, and the residue was dissolved
in DCM (10 mL) and stirred with saturated aqueous KF solution
(10 mL) for 30 min. The mixture was filtered through Celite. The
Celite was washed with DCM, and the combined organic filtrate
was washed with water. The organic fraction was dried, the solvent
evaporated, and the residue purified by chromatography, eluting
with DCM to give product, which was, if necessary, further purified
by chromatography, eluting with 20% EtOAc/petroleum ether, to
give 3-ethyl-1-oxide 6. The yields of the 1-oxides 6 are given in
Table 1.
Alternative Preparation of 3-Ethyl-6-methoxy-1,2,4-benzo-
triazine 1-Oxide (6f). Sodium (45 mg, 1.9 mmol) dissolved in
MeOH (10 mL) was added to a stirred solution of fluoride (250
mg, 1.3 mmol) in MeOH (10 mL), and the solution was stirred at
20 °C for 2 h under N2. The mixture was concentrated, and the
precipitate was filtered off and washed with water to give 1-oxide
6f (254 mg, 95%) as a white solid: mp (EtOAc/petroleum ether)
109-111 °C; spectroscopically identical to 6f prepared above.
Alternative Preparation of 3-Ethyl-7-methoxy-1,2,4-benzo-
triazine 1-Oxide (6g). Sodium (18 mg, 0.8 mmol) dissolved in
MeOH (5 mL) was added to a stirred solution of fluoride (100 mg,
0.5 mmol) in MeOH (5 mL), and the solution was stirred at 20 °C
for 2 h under N2. The mixture was concentrated, and the precipitate
was filtered off and washed with water to give 1-oxide 6g (96 mg,
91%) as a yellow solid: mp (EtOAc/petroleum ether) 98-100 °C;
spectroscopically identical to 6g prepared above.
3-Ethyl-6-methoxy-1,2,4-benzotriazine 1-Oxide (6f). Reaction
of chloride 5f gave (i) starting material 5f (34%) and (ii) 1-oxide
6c (28%) as a white solid: mp (EtOAc/petroleum ether) 109-111
1
°C; H NMR: δ 8.32 (d, J ) 9.5 Hz, 1H), 7.24 (dd, J ) 9.5, 2.6
Hz, 1H), 7.19 (d, J ) 2.6 Hz, 1H), 3.98 (s, 3H), 3.00 (q, J ) 7.6
Hz, 2H), 1.43 (t, J ) 7.6 Hz, 3H). 13C NMR: δ 168.8 (C), 165.3
(C), 150.3 (C), 128.5 (C), 122.9 (CH), 121.7 (CH), 105.8 (CH),
56.2 (CH3), 30.7 (CH2), 12.2 (CH3). Anal. Calcd for C10H11N3O2:
C, 58.53; H, 5.40; N, 20.48. Found: C, 58.64; H, 5.37; N, 20.51%.
3-Ethyl-7-methoxy-1,2,4-benzotriazine 1-Oxide (6g). Reaction
of chloride 5g gave (i) starting material 5g (70%) and (ii) 1-oxide
6g (16%) as a bright yellow solid: mp (EtOAc/petroleum ether)
1
98-100 °C. H NMR: δ 7.88 (d, J ) 9.2 Hz, 1H), 7.72 (d, J )
2.8 Hz, 1H), 7.55 (dd, J ) 9.2, 2.6 Hz, 1H), 3.99 (s, 3H), 3.04 (q,
J ) 7.6 Hz, 2H), 1.44 (t, J ) 7.6 Hz, 3H). 13C NMR: δ 166.0 (C),
160.9 (C), 144.0 (C), 133.8 (C), 129.9 (CH), 128.9 (CH), 97.6 (CH),
56.4 (CH3), 30.5 (CH2), 12.4 (CH3). Anal. Calcd for C10H11N3O2:
C, 58.53; H, 5.40; N, 20.48. Found: C, 58.43; H, 5.42; N, 20.70%.
3-Ethyl-6-methoxy-7-methyl-1,2,4-benzotriazine 1-Oxide (6h).
Reaction of chloride 5h gave (i) starting material 5h (75%) and
(ii) 1-oxide 6h (14%) as an off-white solid: mp (EtOAc/petroleum
ether) 111-113 °C. 1H NMR: δ 8.20 (d, J ) 0.9 Hz, 1H), 7.15 (s,
1H), 4.02 (s, 3H), 3.00 (q, J ) 7.6 Hz, 2H), 2.40 (d, J ) 0.9 Hz,
3H), 1.43 (t, J ) 7.6 Hz, 3H). 13C NMR: δ 167.9 (C), 164.3 (C),
149.3 (C), 134.2 (C), 128.0 (C), 120.3 (CH), 104.4 (CH), 56.4
(CH3), 30.6 (CH2), 17.0 (CH3), 12.3 (CH3). Anal. Calcd for
C11H13N3O2: C, 60.26; H, 5.98; N, 19.17. Found: C, 60.45; H,
5.85; N, 19.09%.
3-Ethyl-7-methoxy-6-methyl-1,2,4-benzotriazine 1-Oxide (6i).
Reaction of chloride 5i gave (i) starting material 5i (76%) and (ii)
1-oxide 6i (14%) as a yellow solid: mp (EtOAc/petroleum ether)
1
89-91 °C. H NMR: δ 7.71 (d, J ) 0.9 Hz, 1H), 7.66 (s, 1H),
4.01 (s, 3H), 3.02 (q, J ) 7.6 Hz, 2H), 2.43 (d, J ) 0.9 Hz, 3H),
1.43 (t, J ) 7.6 Hz, 3H). 13C NMR δ 166.0 (C), 159.9 (C), 143.9
(C), 140.4 (C), 132.4 (C), 128.8 (CH), 96.3 (CH), 56.4 (CH3), 30.5
(CH2), 17.3 (CH3), 12.4 (CH3). Anal. Calcd for C11H13N3O2: C,
60.26; H, 5.98; N, 19.17. Found: C, 59.96; H, 5.83; N, 19.10%.
3-Ethyl-6-fluoro-1,2,4-benzotriazine 1-Oxide (6j). Reaction of
chloride 5j gave 1-oxide 6j (75%) as a white solid: mp (EtOAc/
petroleum ether) 122-124 °C. 1H NMR: δ 8.48 (dd, J ) 9.5, 5.5
Hz, 1H), 7.60 (dd, J ) 8.7, 2.6 Hz, 1H), 7.38-7.44 (m, 1H), 3.04
(q, J ) 7.6 Hz, 2H), 1.43 (t, J ) 7.6 Hz, 3H). 13C NMR: δ 168.6
(q, J ) 175 Hz, C), 165.1 (C), 149.5 (d, J ) 15 Hz, C), 130.5 (C),
123.2 (d, J ) 11 Hz, CH), 120.0 (d, J ) 26 Hz, CH), 112.7 (d, J
Microwave-Assisted Stille Coupling of Chlorides 5a-k.
General Method. Pd(PPh3)4 (0.025 mmol) was added to a N2-
purged, stirred solution of chloride 5 (0.50 mmol) and Et4Sn (0.60
mmol) in MeCN (10 mL) in 100 mL sealed TFM rotors equipped
with a pressure and temperature sensor, as well as a magnetic stirrer.
The reaction tube was placed in a homogeneous microwave
synthesis system, and the system was operated at 140 ( 5 °C,
measured by an internal fiber-optic temperature sensor immersed
in the reaction mixture, for 20 min with a 4 min ramp time. The
6534 J. Org. Chem., Vol. 71, No. 17, 2006

Stille Coupling Reactions of Anticancer Agents
power input was controlled at an average power of 250 W by the
internal temperature sensor. After completion of the reaction, the
solvent was evaporated, and the residue was dissolved in DCM
(10 mL) and stirred with saturated aqueous KF solution (10 mL)
for 30 min. The mixture was filtered through Celite. The Celite
was washed with DCM, and the combined organic filtrate was
washed with water. The organic fraction was dried, the solvent
evaporated, and the residue purified by chromatography, eluting
with DCM to give crude product, which was, if necessary, further
purified by chromatography, eluting with 20% EtOAc/petroleum
ether, to give 3-ethyl-1-oxide 6.
Microwave-Assisted Stille Coupling of Iodides 8a-k. General
Method. Pd(PPh3)4 (0.020 mmol) was added to a N2-purged, stirred
solution of iodide 8 (0.40 mmol) and Et4Sn (0.48 mmol) in MeCN
(10 mL) in 100 mL sealed TFM rotors equipped with a pressure
and temperature sensor, as well as a magnetic stirrer. Reactions
were carried out as described above for chlorides 5.
Preparation of SN29751 (1): 3-Ethyl-6-fluoro-1,2,4-benzo-
triazine 1-Oxide (6j). Nine portions of Pd(PPh3)4 (250 mg, 0.22
mmol) were added to nine N2-purged, stirred solutions of chloride
5j (1.0 g, 5.0 mmol) and Et4Sn (1.2 mL, 6.0 mmol) in MeCN (40
mL) in nine 100 mL sealed TFM rotors. The reaction tubes were
placed in a homogeneous microwave synthesis system, and the
system was operated at 140 ( 5 °C, measured by an internal fiber-
optic temperature sensor immersed in the reaction mixture, for 15
min with a 4 min ramp time. The power input was controlled at an
average power of 220 W by the internal temperature sensor. After
completion of the reaction, the nine reaction mixtures were
combined, and the solvent was evaporated. The residue was
dissolved in DCM (250 mL) and stirred with saturated aqueous
KF solution (250 mL) for 30 min. The mixture was filtered through
Celite. The Celite was washed with DCM, and the combined organic
filtrate was washed with water. The organic fraction was dried,
the solvent evaporated, and the residue purified by chromatography,
eluting with DCM to give 1-oxide 6j (7.30 g, 84%) as an off-white
solid: mp (EtOAc/petroleum ether) 122-124 °C; spectroscopically
identical to 6j prepared above.
fraction was dried, and the solvent was evaporated. The residue
was purified by column chromatography, eluting with a gradient
(0-5%) of MeOH/DCM to give 1-oxide 9 (18.82 g, 76%) as a
pale yellow solid: mp 108-111 °C. 1H NMR: δ 8.33 (d, J ) 9.3
Hz, 1H), 7.21-7.26 (m, 2H), 4.22 (t, J ) 6.4 Hz, 2H), 3.73 (t, J
) 4.6 Hz, 4H), 3.00 (q, J ) 7.6 Hz, 2H), 2.55 (t, J ) 7.0 Hz, 2H),
2.48 (t, J ) 4.6 Hz, 4H), 2.06 (m, 2H), 1.43 (t, J ) 7.6 Hz, 3H).
13C NMR: δ 168.7 (C), 164.6 (C), 150.3 (C), 128.4 (C), 123.1
(CH), 121.6 (CH), 106.3 (CH), 67.3 (CH2), 66.9 (2 × CH2), 55.1
(CH2), 53.7 (2 × CH2), 30.7 (CH2), 26.0 (CH2), 12.2 (CH3). Anal.
Calcd for C16H22N4O3: C, 60.36; H, 6.97; N, 17.60. Found: C,
60.42; H, 7.00; N, 17.40%.
3-Ethyl-6-[3-(4-morpholinyl)propoxy]-1,2,4-benzotriazine 1,4-
Dioxide (1). Hydrogen peroxide (70%; 12.6 mL, ca. 251 mmol)
was added dropwise to a stirred solution of TFAA (35.5 mL, 251
mmol) in DCM (100 mL) at 5 °C (CAUTION: exotherm). The
solution was stirred at 20 °C for 5 min, then cooled to 5 °C, and
added to a solution of 1-oxide 9 (8.0 g, 25.1 mmol) and TFA (9.7
mL, 126 mmol) in DCM (100 mL) at 5 °C. The solution was stirred
at 20 °C for 3 h, cooled to 5 °C, and water (50 mL) was added.
Concentrated NH3 solution (60 mL) was added dropwise to the
vigorously stirred mixture until the mixture was basic, and then
the mixture was stirred for 30 min. The mixture was extracted with
CHCl3 (4 × 100 mL). The combined organic fraction was dried,
and the solvent evaporated. The residue was purified by column
chromatography, eluting with a gradient (0-5%) of MeOH/DCM
to give (i) unreacted starting material 9 (3.63 g, 45%) and (ii) 1,4-
1
dioxide 1 (1.98 g, 24%) as a yellow solid: mp 123-126 °C. H
NMR: δ 8.36 (d, J ) 9.5 Hz, 1H), 7.77 (d, J ) 2.6 Hz, 1H), 7.36
(dd, J ) 9.5, 2.6 Hz, 1H), 4.29 (t, J ) 6.4 Hz, 2H), 3.72 (t, J ) 4.6
Hz, 4H), 3.21 (q, J ) 7.5 Hz, 2H), 2.54 (t, J ) 7.0 Hz, 2H), 2.47
(t, J ) 4.6 Hz, 4H), 2.04-2.10 (m, 2H), 1.44 (t, J ) 7.5 Hz, 3H).
13C NMR: δ 165.1 (C), 157.1 (C), 141.5 (C), 129.6 (C), 124.4
(CH), 123.4 (CH), 98.0 (CH), 68.1 (CH2), 66.9 (2 × CH2), 55.0
(CH2), 53.7 (2 × CH2), 25.9 (CH2), 24.1 (CH2), 9.3 (CH3). Anal.
Calcd for C16H22N4O4: C, 57.47; H, 6.63; N, 16.76. Found: C,
57.17; H, 6.52; N, 16.54%.
3-Ethyl-6-[3-(4-morpholinyl)propoxy]-1,2,4-benzotriazine 1-Ox-
ide (9). A solution of 3-(4-morpholinyl)propanol25 (22.53 g, 155
mmol) in dry THF (70 mL) was added dropwise to a stirred
suspension of NaH (60% dispersion in oil, 6.21 g, 155 mmol) in
dry THF (100 mL) at 20 °C, and the mixture was stirred for 30
min. A solution of fluoride 6j (15.0 g, 77.7 mmol) was added, and
the resulting solution was stirred for 2.5 h. The reaction was cooled
to 0 °C, carefully quenched with water (30 mL), and the solution
was extracted with DCM (4 × 100 mL). The combined organic
Acknowledgment. The authors thank Professor W. A.
Denny for helpful advice and Dr Maruta Boyd for technical
assistance. This work was supported by the US National Cancer
Institute, Grant 1PO1-82566-01A1, and Proacta Therapeutics
Ltd.
Supporting Information Available: General experimental
1
details, data for all compounds, tables of reaction yields, and H
and 13C NMR spectra of new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
(25) Ple, P. A.; Green, T. P.; Hennequin, L. F.; Curwen, J.; Fennell, M.;
Allen, J.; Lambert-van der Brempt, C.; Costello, G. J. Med. Chem. 2004,
47, 871-887.
JO060986G
J. Org. Chem, Vol. 71, No. 17, 2006 6535