New straightforward synthesis of 2-amino-6-methyl-5-(pyridin-4-ylsulfanyl)- 3H-quinazolin-4-one

Article abstract of DOI:10.1080/00397910600978085

The synthesis of 2-amino-6-methyl-5-(pyridin-4-ylsulfanyl)-3H-quinazolin-4- one (1) was studied via three different synthetic routes starting from 4-bromo-5-methylisatin. The best route was established, which is a straightforward route via 2-guanidino-5-b

Full text of DOI:10.1080/00397910600978085

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New Straightforward Synthesis
of2‐Amino‐6‐methyl‐5‐(pyridin‐4‐ylsulfanyl)‐3H‐q
Wei‐Min Chen a b & Shan‐He Wan b
a Department of Medicinal Chemistry, School of Pharmacy, Jinan University,
Guangzhou, China
b Institute of Pharmaceutical Sciences, First Military Medical University,
Guangzhou, China
Version of record first published: 25 Jul 2007.
To cite this article: Wei‐Min Chen & Shan‐He Wan (2007): New Straightforward Synthesis
of2‐Amino‐6‐methyl‐5‐(pyridin‐4‐ylsulfanyl)‐3H‐quinazolin‐4‐one, Synthetic Communications: An International
Journal for Rapid Communication of Synthetic Organic Chemistry, 37:1, 53-61
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Synthetic Communicationsw, 37: 53–61, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910600978085
New Straightforward Synthesis of
2-Amino-6-methyl-5-(pyridin-4-ylsulfanyl)-
3H-quinazolin-4-one
Wei-Min Chen
Department of Medicinal Chemistry, School of Pharmacy, Jinan
University, Guangzhou, China and Institute of Pharmaceutical Sciences,
First Military Medical University, Guangzhou, China
Shan-He Wan
Institute of Pharmaceutical Sciences, First Military Medical University,
Guangzhou, China
Abstract: The synthesis of 2-amino-6-methyl-5-(pyridin-4-ylsulfanyl)-3H-quinazo-
lin-4-one (1) was studied via three different synthetic routes starting from
4-bromo-5-methylisatin. The best route was established, which is a straightforward
route via 2-guanidino-5-bromo-6-methyl-quinazolin-4-one (6) as the key intermediate
and a one-pot synthesis by treatment of compound 6 with 4-mercaptopyridine and
KOH via the Ullmann reaction. When removing the amidino group from 2-guanyl
of compound 6 under strong alkaline conditions, surprisingly we found that 4-mer-
captopyridine could prevent the bromo group on a quinazoline ring from substitution
by a hydroxyl group. Furthermore we found that 4-mercaptopyridine analogues
such as hydroxypyridinones also play a role of protecting agent in such a reaction
system.
Keywords: bromo group, 4-mercaptopyridine, protecting agent, quinazolin-4-one,
synthesis
Received in Japan January 31, 2006
Address correspondence to Wei-Min Chen, School of Pharmacy, Jinan University,
Guangzhou 510632, China. E-mail: twmchen@jnu.edu.cn
53

54
W.-M. Chen and S.-H. Wan
INTRODUCTION
The 2-amino-6-methyl-5-(pyridin-4-ylsulfanyl)-3H-quinazolin-4-one (1)
dihydrochloride, known as Thymitaq or AG337, was designed by protein-
structure-based techniques as a novel nonclassical antifolate.[1,2] Compound 1
[1,2]
was initially synthesized by Webber and coworkers.
Webber’s work
indicated that isatoic anhydride 3 could not be synthesized directly from
isatin (2) through a peroxyacid oxidation. In addition, 4 could not be obtained
from the reaction between anhydride 3 and guanidine. They instead prepared
4 by reacting 2-amino-6-bromo-methylbenzoate with chloroformamidine
hydrochloride.[1,2] After we succeeded in synthesizing 3 by a Baeyer–Villiger
reaction,[3] we undertook a program in the hope of uncovering novel synthetic
routes for synthesis of 1. Three new approaches are delineated (Scheme 1).
Scheme 1. Three routes to 2-amino-6-methyl-5-(pyridin-4-ylsulfany)-3H-quinazo-
lin-4-one.
RESULTS AND DISCUSSION
Route 1: Synthesis of 1 via 2-Amino-5-bromo-3,4-dihydro-6-
methylquinazolin-4-one (4) as Intermediate
We first tried to synthesize 4 via a reaction of 3 and guanidine.[4] A mixture of
anhydride 3 and guanidine carbonate in N,N-dimethylformamide (DMF) was
heated under reflux conditions, affording 4 with a yield of less than 30%.
The isolated products were analyzed, and one of the by-products, 2-amino-5-
hydroxy-6-methyl-3H-quinazolin-4-one (7), was found, which was produced
by a nucleophilic substitution of the bromo group of 4 by a hydroxyl group
as shown in Scheme 2.
The target compound 1 was synthesized via a similar method as
Webber’s.[1,2] We modified the original method (i.e., using K2CO3 instead

New Straightforward Synthesis of Thymitaq
55
Scheme 2. Reaction of 5-bromo-6-methyl-1H-benzo[d][1,3]oxazine-2,4-dione with
guanidine carbonate.
of NaH as base and using ethylene glycol as solvent instead of N,N-dimethyl-
acetamide (DMA)). The modified method improved the yield from 50% to
76%. More important, the modified method is more convenient than
Webber’s method. The reaction need not be carried out in an inert atmosphere.
Route 2: Synthesis of 1 via 2-Guanidino-5-bromo-3,4-dihydro-6-
methylquinazolin-4-one (6) and 2-Amino-5-bromo-3,4-dihydro-6-
methylquinazolin-4-one (4) as Intermediates
Although route 1 can be readily performed to produce 1, the yield of 4
syntheses is not acceptable. We hoped to improve the synthesis of 4. We
reasoned that compound 5 can be easily obtained via oxidation of 2 by
H2O2 and hydrolysis under base condition as reported in Webber’s
method.[1] Treatment of 5 with dicyandiamide in 10% diluted sulfuric acid
under reflux produced compound 6 in high yield (.70%).[5] Cleavage of
the amidino moiety of 6 would afford compound 4, which can then be
converted to the target compound 1. Treatment of 6 with potassium
hydroxide in ethylene glycol[6] produced 2-amino-5-hydroxy-6-methyl-3H-
quinazolin-4-one (7) instead of 4 as expected. In spite of many attempts to
remove the amidino moiety at the 2-position of compound 6 under a variety
of reaction conditions, including microwave irradiation to shorten the
reaction time, the 5-position bromo group was displaced by a hydroxyl group.
In an unexpected experiment, we discovered that compound 4 could be
obtained in good yield when 4-mercaptopyridine was accidentally added
(Scheme 3).
It is therefore clear that 4-mercaptopyridine protected the bromo group
from being substituted by a hydroxyl group under a strong alkaline
condition. We have not found a precedent for this reaction. To find out if
this is a general phenomenon or a special situation, we selected and tested
several 4-mercaptopyridine analogues under the same reaction conditions.
We found that hydroxypyridinones such as 1,2-dimethyl-3-hydroxypyridin-
4-one and 2-methyl-3-hydroxypyridin-4-one also had protective effects. The
best one was 4-mercaptopyridine.
Furthermore, the influence of different reaction times on the 4 syntheses
was investigated in the presence of 2 equivalents of 4-mercaptopyridine. The

56
W.-M. Chen and S.-H. Wan
Scheme 3. Treatment of
4-mercaptopyridine.
6
with KOH in the presence and absence of
formation of 7 was not observed when the reaction time varied from 2 to 24 h.
It demonstrated that 4-mercaptopyridine did play a role of protective agent.
The amount of 4-mercaptopyridine was also studied, and the molar ratio of
6 with 4-mercaptopyridine was tested from 1 : 1 to 1 : 5 and 5 : 1 to 1 : 1; it
was found that the ratio 1 : 2 was the best. We are now conducting research
to find the mechanism of this protective effect by 4-mercaptopyridine.
Route 3: One-Pot Synthesis of 1 via 2-Guanidino-5-bromo-3,4-
dihydro-6-methylquinazolin-4-one (6) as a Key Intermediate
Compound 6 has a poor solubility in most of solvents. It was not dissolved in
common solvents such as methanol, ethanol, chloroform, dichloromethane, or
acetone, but it could be dissolved in polar nonproton solvents such as DMF and
DMSO as well as ethylene glycol at high temperature. Since 4-mercaptopyri-
dine was used in the final step of route 1, it became clear that synthesis of 1 via
6 in one pot was a reasonable approach. Therefore, compound 6 in ethylene
glycol was refluxed for 2 h in the presence of 4-mercaptopyridine, KOH,
CuBr, and Cu2O. The reaction was quenched with water and neutralized.
After filtration and treatment with H2S, compound 1 was obtained with 75%
yield. Compound 1 was then crystallized from 2 equimolar HCl solution and
recrystallized from water, affording white needles. The structure was charac-
1
terized by H NMR, 13C NMR, MS, and elemental analyses and was found
to be a complex of 1 with 2HCl and 2H2O by X-ray crystallography (Fig. 1).
CONCLUSION
The target compound 1 was synthesized via three different synthetic
routes starting from 4-bromo-5-methylisatin. The best route is that via

New Straightforward Synthesis of Thymitaq
57
Figure 1. Crystal structure of compound 1.
compound 6 as a key intermediate and one-pot treatment of the 6 with
4-mercaptopyridine and KOH via Ullmann reaction. We demonstrated
that 4-mercaptopyridine and its analogues could be used to prevent the -
Br on quinazoline ring from being substituted by -OH under strong
alkaline conditions.
EXPERIMENTAL
Melting points were determined with Electrothermer IA9100 and are uncor-
rected. IR spectra were recorded on a Nicolet FT-IR spectrophotometer as
KBr pellets and are expressed as in centimeters21. NMR spectra were
recorded on Varian Unity Inova 500NB (500 MHz) in CDCl3 or DMSO,
and chemical shifts are expressed as in parts per million (ppm). Mass
spectra were obtained on an HP 5988A mass spectrometer (EI) and VG
ZAB-HS mass spectrometer (FAB). Elemental analyses were performed at
Zhongshan University Analyses Center, China.
The solvents were available commercially and were purified according to
conventional methods.

58
W.-M. Chen and S.-H. Wan
6-Amino-2-bromo-3-methyl-benzoic Acid (5)
A mixture of 80 mL of 3 mol/L NaOH and 4-bromo-5-methylisatin 2 (19 g,
80 mmol) was heated at 808C. To the solution was added 18 mL of 30%
H2O2, and the mixture was stirred for 1 h. The mixture was cooled to 58C
and acidified to pH5 with concentrated HCl. The solution was evaporated to
dryness and then added to 300 mL of methanol. The mixture was filtered,
and the filtrate was evaporated to yield a tan solid 5 (18 g, 97.8%). mp:
1
290–2938C (lit.:[1] 290–2948C). H NMR (DMSO-d6): d 2.13 (s, 3H), 4.9
(s, 2H), 6.4 (d, J ¼ 7.8 Hz, 1H), 6.74 (d, J ¼ 7.8 Hz, 1H).
2-Guanidino-5-bromo-3,4-dihydro-6-methylquinazolin-4-one (6)
A mixture of 5 (19.5 g, 70 mmol), 150 mL of H2O, and concentrated H2SO4
was heated to get a clear solution. To the solution was added dicyandiamide
(8.8 g, 100 mmol) under stirring, and the mixture was heated and stirred for
20 min. After cooling, the mixture was added to 50% NaOH to adjust the
pH to 10 and then heated for another 15 min. The mixture was filtered and
washed with water to yield a tan solid 6 (14.8 g, 71.4%). Because of poor solu-
bility of the product in organic solvents, crystallization of 6 could not be done
and 1H NMR of it could not be obtained. MS(EI); 295, 297 (12), 211, 213 (70),
185, 187 (100), 132 (33), 106 (44), 77 (40).
2-Amino-5-bromo-3,4-dihydro-6-methylquinazolin-4-one (4) from 6
A mixture of 6 (20.0 g, 68 mmol), KOH (30.0 g, 536 mmol), and 4-mercapto-
pyridine (15 g, 135 mmol) in ethylene glycol (200 mL) was refluxed for 2 h.
After cooling, the mixture was diluted with water (800 mL) and neutralized
with concentrated HCl. The mixture was filtered and washed with water to
give a tan solid 4 (13 g, 75.3%). mp . 3008C. IR (KBr): 3320, 3177, 2966,
1
1679, 1637, 1567, 1293, 1110, 814, 625 cm21. H NMR (DMSO-d6): 2.3
(3H, s), 6.4 (bs, 2H), 7.05 (d, J ¼ 8.3 Hz, 1H), 7.45 (d, J ¼ 8.0 Hz, 1H), 11
(bs, 1H). MS (EI: m/z, %): 253, 255 (50), 211, 213 (25), 175 (27), 133
(20), 104 (35), 77 (60), 44 (100).
One-Pot Synthesis of 2-Amino-6-methyl-5-(pyridin-4-ylsulfanyl)-
3H-quinazolin-4-one (1) from 6
A mixture of 6 (20.0 g, 68 mmol), KOH (30.0 g, 536 mmol), and 4-mercapto-
pyridine (15 g, 135 mmol) in ethylene glycol (200 mL) was refluxed for 2 h.
After cooling, the mixture was added to CuBr (5.0 g, 35 mmol) and Cu2O
(5.0 g, 35 mmol) and refluxed for another 2 h. The mixture was cooled to

New Straightforward Synthesis of Thymitaq
59
room temperature and filtered. The filtrate was diluted with water (20 mL) and
neutralized with concentrated HCl. The mixture was filtered and washed with
water to give a solid. To the resulting solid was added 250 mL of H2S/MeOH
solution (20 g/L). The dark mixture was stirred for 1 h. The precipitated CuS
was removed by filtration, and the methanolic filtrate was evaporated. The
solid was washed with CH2Cl2, followed by ethyl ether, and finally boiling
isopropanol to yield a tan solid (14.5 g, 75%). mp: 301–3028C (lit.:[1] 301–
3028C). IR (KBr): 3325, 3149, 2762, 1672, 1574, 1468, 1307, 1222, 800,
1
709, 484 cm21. H NMR (DMSO-d6) 2.31 (s, 3H), 6.38 (bs, 2H), 6.86 (bs,
2H), 7.27 (d, J ¼ 8.4 Hz, 1H), 7.58 (d, J ¼ 8.5 Hz, 1H), 8.53 (bs, 2H),
10.82 (bs, 1H). MS (FAB): 285 (M þ 1,100%).
Crystallization of 1 and Preparation of Its Hydrochloride
To compound 1 (1.6 g, 5.6 mmol) in H2O (1.46 mL) was added concentrated
HCl (0.94 mL, 11.3 mmol). The mixture was heated to dissolve completely.
The solution was cooled to room temperature, and white needle crystals
were obtained. A sample was redissolved in water to recrystallize and dried
in vacuum. White crystal 1 (1.65 g, 75%) was obtained. Single crystals
were grown from aqueous solution. mp: 3208C (decompose). IR(KBr):
3336, 3140, 1662, 1581, 1467, 1413, 1362, 1221, 835, 804, 712, 669,
487 cm21 1H NMR (500 MHz, DMSO-d6) d 2.45 (s, 3H, -CH3), 7.52
.
(d, J ¼ 7.0 Hz, 2H, pyridyl), 7.72 (d, J ¼ 8.5 Hz, 1H, benzene), 7.92
(d, J ¼ 8.5 Hz, 1H, benzene), 8.51 (d, J ¼ 7.0 Hz, 2H, pyridyl), 8.54
(bs, ammonium salt). 13C NMR (CDCl3) d 20.6, 95.3, 116.9, 120.5, 121.6,
125.0, 137.9, 139.9, 141.4, 150.8, 157.4, 162.2. MS (FAB): 285 (M þ 1,
.
.
100%). Anal. calcd. for C14H12N4OS 2HCl 2H2O: C, 42.75; H, 4.61; N,
14.25; S, 8.15. Found: C, 42.84; H, 4.69; N, 14.48; S, 8.09.
5-Bromo-6-methyl-1H-benzo[d][1,3]oxazine-2,4-dione (3)[3,7]
A suspension of 2 (9.6 g, 40 mmol) in glacial acetic acid (40 mL) and concen-
trated sulfuric acid (0.3 mL) was stirred and added dropwise within 30 min
with newly prepared peracetic acid [acetic anhydride (8 mL) with 30%
H2O2 (9 mL)]. The mixture was kept at 60–708C for 4 h. After cooling to
room temperature, the precipitate was filtered off and washed with water,
5% NaHCO3, and water. After recrystallization with absolute ethanol, a
1
yellowish crystal 3 was obtained (6.5 g, 70%). mp. 270–2738C. H NMR
(DMSO-d6) d 2.38 (s, 3H, -CH3), 7.05 (d, J ¼ 8.4 Hz, 1H), 7.65
(d, J ¼ 8.4 Hz, 1H), 11.75 (s, 1H, -NH); MS (m/z, %): 255 (18.8), 257
(18.8), 239, (4), 241 (4), 211 (67), 213 (67), 184 (20.8), 186 (20.8), 133
(8.3), 104 (20.8), 77 (27.1), 44 (100).

60
W.-M. Chen and S.-H. Wan
2-Amino-5-bromo-6-methyl-3H-quinazolin-4-one (4)
A mixture of 3 (3 g, 16 mmol) and guanidine carbonate (4.3 g, 23 mmol) in
DMF (40 mL) was heated under reflux for 24 h. The reaction mixture was
cooled to ambient temperature, guanidine carbonate (0.7 g, 5.8 mmol) was
added, and heating was continued for an additional 7 h. Most of the DMF
was removed under vacuum, the residue was added to H2O (80 mL), and a pre-
cipitate was produced. The mixture was filtered and washed with water. The
solid was recrystallized from DMSO–water to yield a light grey solid (1.3 g,
32%).
The solid is not very pure; HPLC analysis and LC-MS found it contain a
side product of 2-amino-5-hydroxy-6-methyl-3H-quinazolin-4-one (7).
1
MS (m/z %): 191 (100), 192 (11). H NMR (500 MHz, DMSO-d6): 2.35
(s, 3H, -CH3), 2.47 (s, 2H, -NH2), 6.35 (bs, 1H, -OH), 7.30 (d, J ¼ 8.5 Hz,
2H, benzene), 7.60 (d, J ¼ 8.5 Hz, 2H, benzene), 11.0 (br, 1H, -NH-).
Synthesis of 2-Amino-6-methyl-5-(pyridin-4-ylsulfanyl)-3H-
quinazolin-4-one (1) from 4
A mixture of 4 (8.0 g, 31 mmol), K2CO3 (4.35 g, 31 mmol), 4-mercaptopyri-
dine (7.0 g, 63 mmol), CuBr (2.0 g, 14 mmol), and Cu2O (2.0 g, 14 mmol) in
ethylene glycol (100 mL) was refluxed for 2 h. The mixture was cooled to
room temperature and filtered. The filtrate was diluted with water (200 mL)
and neutralized with concentrated HCl. The mixture was filtered and
washed with water to give a solid. To the resulting solid was added 250 mL
of H2S/MeOH solution (20 g/L). The dark mixture was stirred for 1 h. The
precipitated CuS was removed by filtration, and the methanolic filtrate was
evaporated. The solid was dissolved in H2O (200 mL), and the pH was
adjusted to 7 with concentrated NH3.H2O. The mixture was filtered, washed
with water, and dried in vacuum to yield, tan solid (6.7 g, 76%).
X-ray Single-Crystal Structure Analyses of Compound 1
The structure of 1 was determined by X-ray crystallography with a crystal that
measured 0.58 ꢀ 0.44 ꢀ 0.32 mm. Diffraction measurements were made on a
Siemens P4 diffractometer with graphite monochromated MoKa
˚
(l ¼ 0.71073 A) radiation. Preliminary indications of the unit cell based on
randomly selected reflections revealed orthorhombic symmetry with the
˚
˚
following lattice parameters: a ¼ 10.0980(10) A, b ¼ 8.7490(10) A,
˚
c ¼ 20.481(3) A, with a ¼ g ¼ 90.08, b ¼ 101.600(10)8. The space group
was P2/n, Z ¼ 4 with one molecule of composition C14H18Cl2N4O3S. The
calculated density was 1.474 g/cm3. There were 4292 reflections collected
with u ranging from 2.03 to 25.008; of those reflections, 3119 with

New Straightforward Synthesis of Thymitaq
61
I . 2s(I) were observed. The structure was solved by SHELXTL 5.03. The
crystal structure of 1 has been deposited at the Cambridge Crystallography
Data Centre (CCDC 606437).
ACKNOWLEDGMENT
This work was partially supported by grants from the Guangdong Provincial
Natural Science Foundation (No. 994078) and Foundation of Jinan University.
We are grateful to Kai-Bei Yu of Chengdu Institute of Organic Chemistry for
performing the X-ray crystallographic analysis and Zhongshan University
Analyses Center for NMR, MS, and elemental analyses.
REFERENCES
1. Webber, S. E.; Bleckman, T. M.; Attard, J.; Deal, J. G.; Kathardekar, V.;
Welsh, K. M.; Webber, S.; Janson, C. A.; Matthews, D. A.; Smith, W. W.;
Freer, S. T.; Jordan, S. R.; Bacquet, R. J.; Howland, E. F.; Booth, C. L. J.;
Ward, R. W.; Hermann, S. M.; White, J.; Morse, C. A.; Hilliard, J. A.;
Bartlett, C. A. Design of thymidylate synthase inhibitors using protein crystal struc-
tures: The synthesis and biological evaluation of a novel class of 5-substituted qui-
nazolinones. J. Med. Chem. 1993, 36, 733–746.
2. Webber, S. E.; Bleckman, T. M.; Attard, J.; Jones, T. R.; Varney, M. D. Antiproli-
ferative quinazolines. U.S. Patent 5430148, 1995; Chem. Abstr. 1994, 120, 164214.
3. Chen, W. M.; Zeng, L. M. Baeyer–Villiger oxidation of 4-bromo-5-methylisatin.
Chem. J. Chin. Universities 1999, 20, 421–423 (in Chinese).
4. Singh, S. K.; Govindan, M.; Hynes, J. B. Synthesis of 5-trifluoromethyl-5,8-didea-
zafolic acid and 5-trifluoromethyl-5,8-dideazaisofolic acid. J. Heterocyclic. Chem.
1990, 27, 2101–2105.
5. Han, G. D.; Zhao, S. W.; Li, S. W. (Eds.) Handbook of Organic Preparation
(Volume II); Chemistry Industry Press: Beijing, 1977; p. 305 (in Chinese).
6. Grout, R. J.; Partridge, M. W. Cyclic amidines, part X: 2-aminoquinazoline deriva-
tives. J. Chem. Soc. 1960, 3540–3545.
7. Reissenweber, G.; Mangold, D. Oxidation of isatins to isatoic anhydrides and
2,3-dioxo-1,4-benzoxazines. Angew. Chem., Int. Ed. Engl. 1980, 19, 222–223.