Pharmaceutical Chemistry Journal, Vol. 45, No. 12, March, 2012 (Russian Original Vol. 45, No. 12, December, 2011)
SYNTHESIS AND ANTIVIRAL ACTIVITY OF FLUORINE-CONTAINING 4-ARYLAMINOQUINAZOLINES G. N. Lipunova,1,* E. V. Nosova,2 A. A. Laeva,2 and V. N. Charushin1 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 45, No. 12, pp. 7 – 9, December, 2011. Original article submitted May 24, 2010.
2-Methylthio-4-phenylamino-6,7,8-trifluoro-3H-quinazolin-4-one was synthesized by means of intramolecular cyclizations of S-methyl-N-(tetrafluorobenzoyl)isothiourea followed by a reaction with POCl3 and nucleophilic substitution at the 4-position. The reactions of the synthesized compound with amines proceed, depending on their nature, via substitution of either the F(7) atom or the SMe fragment in the 2-position. The antiviral activity of the obtained 6,7,8-trifluoro-2-ethylthioquinazolin-4-ones was investigated using monkeypox virus, smallpox vaccine, and ectromelia virus. It is shown that fluorinated quinazoline derivatives have good prospects in the search for new active substances. Key words: 2-methylthio-4-phenylamino-6,7,8-trifluoro-3H-quinazolin-4-one, 6,7,8-trifluoro-2-ethylthioquinazolin-4-ones, synthesis, antiviral activity.
The development of new antiviral drugs remains a critical problem. Viral infections include vectors of dangerous viral infections such as natural pox virus, which causes serious human disease with lethality up to 40 – 60%. Therefore, highly effective antiviral drugs that exhibit high activity, low toxicity, and prolonged action against human pathogenic orthopox viruses are needed. Many quinazoline derivatives exhibit a broad spectrum of biological activity. Thus, 4-arylaminoquinazolines are inhibitors of tyrosine-kinase [1 – 4] and adenosine-kinase [5] receptors. Certain 4-arylaminoquinazolines exhibit antitumor activity [6 – 9]. Derivatives of 8-fluoroquinazolinone are active against human cytomegalovirus [10]. 4-Anilinoquinazolines are neuraminidase pox virus inhibitors [11]. Therefore, it seemed interesting to prepare fluorine-containing 4-arylaminoquinazolines in order to search in their series for compounds with antiviral activity. We developed earlier convenient methods for synthesizing 6,7,8-trifluoro-2-ethylthio-4-arylaminoquinazolines (I) [12]. Treatment of 6,7,8-trifluoro-2-ethylsulfanylquinazolin4-ones with POCl3 and subsequent heating of the 4-chloroquinazolines with amines in CH3CN produced 1a – e. It seemed interesting to study their antiviral activity. 1 2 *
HN F
R N
F
N F
SEt
I
R: Ph (a), 2-ClC6H4 (b), 3,4-F2C6H3 (c); benzyl (d); 5-tert-butylisoxazol-3-yl (e).
2-Methylthio-4-phenylamino-6,7,8-trifluoro-3H-quinazo lin-4-one (IV) was synthesized in order to vary the substituents in the 2- and 7-positions (Scheme 1). The reaction of tetrafluorobenzoylchloride with S-methylisothiourea hydroiodide at room temperature in CHCl2 in the presence of Et3N gave S-methyl-N-(tetrafluorobenzoyl)isothiourea (II), which underwent intramolecular cyclization to form the quinazolinone (III) upon refluxing in DMF. Compound III was treated with POCl3. The resulting 4-chloro quinazoline derivative underwent nucleophilic substitution at the 4-position to afford substituted 4-phenylaminoquinazoline IV. Reaction of IV with amines depended on their nature and occurred with substitution of F(7) to form Va and Vb or of the SMe fragment in the 2-position to form VIa and -b. We noted earlier that the direction of the nucleophilic substitution (at the 2- and 7-positions) in fluorine-containing 1,3-benzothiazin-4-ones varied as a function of the nature of the amine [13].
I. Ya. Postovskii Institute of Organic Synthesis, Ural Branch, Russian Academy of Sciences, Yekaterinburg, 620219, Russia. B. N. Yeltsin Ural Federal University, Yekaterinburg, 620002, Russia; e-mail:
[email protected] e-mail:
[email protected].
709 0091-150X/12/4512-0709 © 2012 Springer Science+Business Media, Inc.
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G. N. Lipunova et al. Scheme 1 H2N
O
SMe
O
HI
F
Cl
F
F
NH NEt 3
F
NH N H
F
SMe
F
F
F II R HN R O
DMF, t
F N
HN 2
F
HN 1) POCl 3
NH
F
1
F
PhNH2 SMe 2)
N
HN
F
III
R
IV
R
N
N R
SMe
N
1
N
F
F
R
2
SMe
F V
1
2
HN F
N N
F F VI
R
N R
1
2
V: NR1R2 = pyrrolidin (a), 3-methylpiperidin-1-yl (b). VI: NR1R2 = morpholin-4-yl (a), 4-(morpholin-4-yl)propylamino (b).
EXPERIMENTAL CHEMICAL PART PMR spectra were recorded in DMSO-d6 with TMS internal standard on a Bruker DRX-400 spectrometer at operating frequency 400.13 MHz. Mass spectra were recorded in a Shimadzu LCMS-2010 liquid chromatograph-mass spectrometer. Elemental analyses agreed with the empirical formulas. 2-Methyl-1-(2,3,4,5-tetrafluorobenzoyl)isothiourea (II). A solution of S-methylisothiourea hydroiodide (3.0 g, 13 mmol) in anhydrous CH2Cl2 (30 mL) was treated with NEt3 (3.6 mL, 25.5 mmol) and then a solution of tetrafluorobenzoic acid chloride (13 mmol) in toluene
TABLE 1. Cytotoxic and Antiviral Activity of 2-Ethylthio-4anilino-6,7,8-trifluoroquinazolines (Ia – c) Compound
Ib
Ia
Ic
Ar
Orthopox viruses
2-ClC6H4 smallpox vaccine monkeypox virus ectromelia C6H5 smallpox vaccine monkeypox virus ectromelia 3.4-F2C6H3 smallpox vaccine monkeypox virus ectromelia
CTA50, mg/mL
IC50, mg/mL
SI
> 100
1.44
> 69.4
0.15
> 666.7
1.49 0.05
> 67.1 > 2000
< 0.05
> 2000
0.05 0.09
> 2000 85.4
< 0.05
> 153.8
0.11
69.9
> 100
7.69
(2.5 mL), stirred at room temperature for 1 d, and evaporated to one fourth the volume. The colorless precipitate of substituted isothiourea II was filtered off, washed with H2O (40 mL), dried, and recrystallized from EtOH. Yield 2.84 g (82%), mp 113 – 115°C. PMR spectrum (DMSO-d6, d, ppm): 2.46 (s, 3H, CH3), 7.73 (m, 1H, C6HF4), 9.43 (br.s, 2H, NH). C9H6N2SOF4. 2-Methylthio-6,7,8-trifluoro-3H-quinazolin-4-one (III). Isothiourea II (1.5 g, 5.6 mmol) was treated with anhydrous DMF (6 mL), refluxed for 4 h, and cooled. The resulting precipitate was filtered off and recrystallized from DMSO. Yield 1.07 g (78%), mp 218 – 220°C. PMR spectrum (DMSO-d6, d, ppm, J/Hz): 2.50 (s, 3H, CH3), 7.73 [ddd, 1H, 3J = 10.1, 4J = 7.7, 5J = 2.1, H(5)], 12.4 (br.s, 1H, NH). C9H5N2OSF3. 6,7,8-Trifluoro-2-methylthio-4-(phenylamino)quinazoline (IV). Quinazolinone III (1 g, 4 mmol) was treated with POCl3 (5 mL), refluxed for 1.5 h, cooled, and poured onto ice. The precipitate of 4-chloroquinazoline was filtered off, dried, treated with anhydrous CH3CN (5 mL) and aniline (0.37 mL, 4 mmol), refluxed for 3 h, and cooled. The precipitate of IV was filtered off, washed with H2O, and recrystallized from EtOH. Yield 0.89 g (69%), mp 200 – 202°C. PMR spectrum (DMSO-d6, d, ppm, J/Hz): 2.50 (s, 3H, CH3), 7.13 (m, 1H, Ph), 7.36 (m, 2H, Ph), 7.76 (m, 2H, Ph), 8.41 [ddd, 1H, J = 11.0, 7.5, 2.1, H(5)], 9.7 (br.s, 1H, NH). Mass spectrum [chemical ionization (CI), m/z, Irel, %]: 322 (100) [M + H]+. C15H10N3SF3. 2-Methylthio-7-(pyrrolidin-1-yl)-6,8-difluoro-4-(phenylamino)quinazoline (Va). A solution of quinazoline IV (0.6 g, 1.9 mmol) in anhydrous DMF (4 mL) was treated with pyrrolidine (0.6 mL, 8.0 mmol), refluxed for 3 h, and
Synthesis and Antiviral Activity of Fluorine-Containing 4-Arylaminoquinazolines
cooled. The resulting precipitate was filtered off and recrystallized from DMSO. Yield 0.54 g (72%), mp 220 – 222°C. PMR spectrum (DMSO-d6, d, ppm, J/Hz): 1.90 [m, 4H (CH3)2], 2.49 (s, 3H, CH3), 4.05 [m, 4H, N(CH2)2], 7.12 (m, 1H, Ph), 7.39 (m, 2H, Ph), 7.79 (m, 2H, Ph), 8.10 [d, 1H, 3J = 9.6, H(5)], 9.5 (br.s, 1H, NH). Mass spectrum (CI, m/z, Irel, %): 373 (100) [M + H]+. C19H18N4SF2. Compounds Vb, VIa, and VIb were synthesized analogously with reaction times of 2 h, 3 h, and 5 h, respectively. 6,8-Difluoro-2-methylthio-7-(3-methylpiperidin-1-yl)4-phenylaminoquinazoline (Vb). Yield 60%, mp 101 – 102°C. PMR spectrum (DMSO-d6, d, ppm, J/Hz): 0.91 (d, 3H, J = 6.6, CH3), 1.15 (m, 1H, CH), 1.74 (m, 4H, 2CH2), 2.50 (s, 3H, SCH3), 2.78 (m, 1H, CH), 3.08 (m, 1H, CH), 3.37 (m, 2H, CH2), 7.14 (m, 1H, Ph), 7.40 (m, 2H, Ph), 7.79 (m, 2H, Ph), 8.18 [m, 1H, H(5)], 9.7 (br.s, 1H, NH). Mass spectrum (CI, m/z, Irel, %): 401 (100) [M + H]+, 384 (22), 347 (87). C21H22N4SF2. 2-(Morpholin-4-yl)-6,7,8-trifluoro-4-(phenylamino)quinazoline (VIa). Yield 68%, mp 142 – 144°C. PMR spectrum (DMSO-d6, d, ppm): 3.32 [m, 4H, N(CH2)2], 3.75 [m, 4H, O(CH2)2], 7.08 (m, 1H, Ph), 7.33 (m, 2H, Ph), 7.79 (m, 2H, Ph), 8.12 [m, 1H, H(5)], 9.5 (br.s, 1H, NH). Mass spectrum (CI, m/z, Irel, %): 389 (100) [M + H]+, 348 (21), 347 (93). C18H15N4OF3. 2-[4-(Morpholin-4-yl)propylamino]-6,7,8-trifluoro-4phenylaminoquinazoline (VIb). Yield 64%, mp 201 – 203°C. PMR spectrum (DMSO-d6, d, ppm, J/Hz): 1.75 (m, 2H, CH2), 2.39 [m, 4H, (CH2)2], 2.49 (s, 3H, CH3), 3.33 (m, 2H, NCH2), 3.50 (m, 2H, NCH2), 3.58 [m, 4H, O(CH2)2], 6.41 (br.s, 1H, NH), 7.12 (m, 1H, Ph), 7.38 (m, 2H, Ph), 7.78 (m, 2H, Ph), 8.11 [d, 1H, 3J = 8.3, H(5)], 9.49 (br.s, 1H, NH). Mass spectrum (CI, m/z): 446 (100) [M + H]+, 347 (20), 365 (15), 244 (37). C22H25N5OS. EXPERIMENTAL BIOLOGICAL PART Tests were conducted on Vero cell culture for monkeypox virus (strain Zair 599), smallpox virus vaccine (strain LIVP used for vaccination of the public), and ectromelia virus.3 Vero cell culture was grown in wells of flat-bottomed 96-well plates. Tested compounds (serial dilutions) and the corresponding virus were added to the culture medium. After incubation for 3 – 5 d, the cell monolayer was stained with the vital neutral red dye. The excess of dye was removed and rinsed away. Lysing solution was added. The amount of dye incorporated into the cell monolayer was calculated on a spectrophotometer at 490 nm. The controls were plate wells without virus (compound toxicity) and without virus and compound (cell culture control). This method enabled an assessment of the capability of the tested com3
We thank E. F. Belanov (SSC VB Vector, Novosibirsk) for performing the biological tests.
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pounds to prevent virus reproduction and its distribution from cell to cell. With this, the cells did not die and retained the ability to phagocytize neutral red. The selectivity index (SI) was calculated from the ratio of cytotoxic activity (CTA50, mg/mL) to antiviral activity (IC50, mg/mL) [14, 15]. RESULTS AND DISCUSSION It was found that compounds Ia – c exhibited pronounced antiviral activity (Table 1). The IC50 value for Id was close to the CTA50 value, i.e., this compound had a SI of about 1. The high SI values for Ia – c (³ 10.0) indicated that the search for new active compounds among fluorine-containing quinazoline derivatives is promising. Compounds Ia – c can be considered to be promising compounds for creating drugs to treat and prevent diseases caused by orthopox viruses that are pathogenic for man and animals. ACKNOWLEDGMENTS The work was supported financially by State Contract GK-02.740.11.0260 and a Scientific Schools Grant NSh-65261.2010.3. REFERENCES 1. R. H. Bradbury, L. F. A. Hennequin, and J. G. Kettle, PCT Int. Appl. (2005), WO2005051923; Chem. Abstr., 143, 43897 (2005). 2. J. W. Kin, S. Y. Kim, Y.-G. Ahn, et al., PCT Int. Appl. (2007), WO2007055513; Chem. Abstr., 146, 521811 (2007). 3. F. Himmelsbach and B. Jung, PCT Int. Appl. (2007), WO2007023073; Chem. Abstr., 146, 295946 (2007). 4. R. H. Bradbury, L. F. A. Hennequin, and B. C. Barlaam, PCT Int. Appl. (2005), WO2005028470; Chem. Abstr., 142, 336338 (2005). 5. K. Gomes Franchini, M. Abdalla Saad, R. Ritter Neto, et al., PCT Int. Appl. (2005), WO2005085213; Chem. Abstr., 143, 306332 (2005). 6. L. F. A. Hennequin, J. G. Kettle, M. Pass, and R. H. Bradbury, PCT Int. Appl. (2003), WO2003040109; Chem. Abstr., 138, 385442 (2003). 7. L. F. A. Hennequin, Pat. Publ. No. US 2005085465 A1; Chem. Abstr., 142, (2005); 139, 164806. 8. P. Sun, PCT Int. Appl. (2006), WO2006081741; Chem. Abstr., 145, 211063 (2006). 9. F. Solca, A. Amelsberg, G. Stehle, et al., PCT Int. Appl. (2007), WO2007054550; Chem. Abstr., 146, 514720 (2007). 10. T. Wunberg, J. Baumeister, U. Betz, et al., PCT Int. Appl. (2004), WO200496778; Chem. Abstr., 141, 410947g (2004). 11. H. Yang, S. K. Kim, M. Kim, P. A. Reche, et al., J. Clin. Invest., 115, 379 – 385 (2005). 12. A. A. Layeva, E. V. Nosova, G. N. Lipunova, et al., J. Fluorine Chem., 128, 748 – 754 (2007). 13. E. V. Nosova, G. N. Lipunova, A. A. Laeva, and V. N. Charushin, Zh. Org. Khim., 42(10), 1555 – 1561 (2006). 14. R. O. Baker, M. Bray, and J. W. Huggins, Antiviral Res., 57, 13 – 23 (2003). 15. J. Paragas, C. A. Whitehouse, and T. P. Endy, Antiviral Res., 62, 21 – 25 (2004).
