Synthesis and in vitro cytotoxicity evaluation of some fluorinated hexahydropyrimidine derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.12.022

A series of trifluoromethylated hexahydropyrimidine and tetrahydropyrimidine derivatives were synthesized and their in vitro cytotoxic activities were determined in colon cancer cell line (COLO 320 HSR). Compounds 4f, 4g, 4k, 5, and 7 proved to be the most active in this series of compounds. They represent promising new leads for the development of highly potent and selective anticancer compounds. All the compounds are lipophilic due to the trifluoromethyl group, and are thus expected to penetrate the membrane in appreciable concentration.

Full text of DOI:10.1016/j.bmcl.2010.12.022

Bioorganic & Medicinal Chemistry Letters 21 (2011) 989–992
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro cytotoxicity evaluation of some fluorinated
hexahydropyrimidine derivatives
Oluropo C. Agbaje a, Olugbeminiyi O. Fadeyi a, S. Adamson Fadeyi b, Lewis. E. Myles b, Cosmas O. Okoro a,
⇑
a Department of Chemistry, Tennessee State University, 3500 John A. Merritt Blvd., Nashville, TN 37209-1561, USA
b Department of Biological Sciences, Tennessee State University, 3500 John A. Merritt Blvd., Nashville, TN 37209-1561, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of trifluoromethylated hexahydropyrimidine and tetrahydropyrimidine derivatives were synthe-
sized and their in vitro cytotoxic activities were determined in colon cancer cell line (COLO 320 HSR).
Compounds 4f, 4g, 4k, 5, and 7 proved to be the most active in this series of compounds. They represent
promising new leads for the development of highly potent and selective anticancer compounds. All the
compounds are lipophilic due to the trifluoromethyl group, and are thus expected to penetrate the mem-
brane in appreciable concentration.
Received 7 October 2010
Revised 2 December 2010
Accepted 7 December 2010
Available online 10 December 2010
Keywords:
In vitro cytotoxicity
Synthesis
Ó 2010 Elsevier Ltd. All rights reserved.
Hexahydropyrimidine
Tetrahydropyrimidine
Trifluoromethyl
Stereogenic
Polyphosphate ester
Colon cancer
The unique properties of the trifluoromethyl group, such as its
strong electron-withdrawing character, lipophilicity, and meta-
bolic stability have been exploited in the design of novel targets
for pharmaceutical, agrochemical and material science indus-
tries.1,2 In particular, fluorine-building blocks are important be-
cause of their extensive use in the synthesis of drugs covering a
wide variety of therapeutic areas. A fluorine atom is similar in size
to a hydrogen atom. Thus, the replacement of hydrogen by fluorine
is expected not to cause any significant change in molecular geom-
etry and shape.3 The high electronegativity of fluorine has a strong
effect on the electronic properties of the basic molecules. On a
molecular level this allows for the inhibition of some metabolic
pathways, including the modulation of membrane permeability,
as well as electrostatic interactions with the target site.4
easy methods for the synthesis of trifluoromethyl ‘carrier reagents’.5
The most efficient, mild and versatile nucleophilic trifluoromethy-
lating reagent is the Ruppert–Prakash reagent (trifluoromethyl–tri-
methylsilane), which upon initiation with fluoride ions, delivers the
trifluoromethyl group to electrophilic substrates.6 The trifluoro-
methyl group has been successfully used as a probe for the hydro-
phobic binding site in human thymidylate synthase.7
The multifunctionalized dihydropyrimidine and hexahydropyr-
imidine scaffolds are one of the most commonly encountered het-
erocycles in medicinal chemistry. They exhibit diverse pharmaco
logical properties, such as antiviral, antitumor, antibacterial and
antiinflammatory activities.8
Dihydropyrimidine, monastrol and its fused bicyclic core analog
dimethylenanstron are the first small molecule inhibitor of the mi-
totic motor Eg5 (kinesin spindle protein, KSP), and are considered
as a new lead in the development of anticancer agents.9 Hexahy-
dropyrimidine nucleus is also present in a number of alkaloids,
such as tetraponerines, verbamethine and verbametrine.10
In continuation of our search for biologically active fluorine-
containing compounds, we report in this letter the polyphosphoric
ester (PPE) promoted synthesis and preliminary in vitro cytotoxic
activity of fluorinated hexahydropyrimidine and tetrahydropyrim-
idine derivatives.
From a physiological standpoint, better bioavailability and en-
hanced selectivity for the target site can be achieved.
In addition, a much lower dose of a fluorinated drug is often
needed in some cases, compared to the unfluorinated ones. We are
particularly interested in trifluoromethylated pharmaceuticals,
since they are present in many commercially available drugs.
Although, several trifluoromethyl building blocks are commercially
available, many of them are expensive. Thus, several research labo-
ratories, including ours are engaged in the investigation of new and
In furtherance of our interest in biologically active fluorine-con-
taining compounds, herein we report the synthesis and anticancer
activity of a series of fluorinated hexahydropyrimidine derivatives.
⇑
Corresponding author. Tel.: +1 615 963 5332; fax: +1 615 963 5326.
E-mail address: cokoro@tnstate.edu (C.O. Okoro).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.022

990
O. C. Agbaje et al. / Bioorg. Med. Chem. Lett. 21 (2011) 989–992
Table 1
Synthesis of fluorinated hexahydropyrimidine and in vitro anticancer activity on colon cancer cell line
ArCHO
R
F3C OH
HN
2
PPE
THF, reflux
O
O
NH2
+
O
R1
CF3
H2N
S
N
H
S
Ar
3
4a-q
Entrya
1
Ar
R
Product
Yield (%)b
80
IC50
75
(l
M)c(COLO 320)
3,4-(C–CH2–O)-C6H3
OEt
4a
2
3
4
5
6
3,4-(C–CH2–O)-C6H3
3-(CH)-C6H4
4b
84
82
75
87
88
62.5
72
S
S
4c
4d
4e
4f
4-(F)-C5H4
51
O
S
4-(F)-C5H4
>100
4-(OMe)-C6H4
11.5
9.3
63
O
7
8
4-(F)-C5H4
C6H5
4g
4h
83
92
O
S
9
4-(OMe)-C6H4
2-(Br)-C6H4
4i
4j
89
78
75
73
10
OEt
11
3,4-(O–CH2–O)-C6FH3
4k
80
9.9
12
13
3-(OH)-C6H4
4l
78
67
>100
O
O
3,4-(O–CH2–O)-C6H3
4m
67
14
15
16
17
4-(F)C6H4
OEt
4n
4o
4p
4q
81
95
93
68
75
>100
>100
>100
3-(OH) C6H4
3-(OH) C6H4
3-(CN)-C6H4
OMe
OEt
OEt
a
b
c
All reactions proceeded to complete conversion.
Yield after chromatography or recrystallization.
IC50 (l
M) values obtained from Alamarblue™ assays with tested cancer cell lines; means obtained from three independent experiments performed in triplicate.15
We initially examined the reaction of trifluoroacetoacetate 1,
appropriate aldehyde 2 and thiourea 3 in ethanol with a catalytic
amount of HCl at reflux temperature.11 Unfortunately, the reaction
was found to occur only when 3-hydroxy benzaldehyde (entries 15
and 16) is used as the aldehyde, giving a low yield of the desired
hexahydropyrimidine product. No reaction was observed when
other aldehyde derivatives were used. This led us to Kappe’s previ-
ous work, where polyphosphate ester (PPE) was proven to be an
efficient reagent in the synthesis of dihydropyrimidine deriva-
tives.12 The PPE was prepared according to Lakshmikantham’s pro-
cedure.13 Utilizing a catalytic amount of the prepared PPE, the
synthesis of the trifluoromethylated hexahydropyrimidine deriva-
tives were achieved in a one pot condensation of 1:1:1.5 ratios of
appropriate aldehyde 2, ethyl trifluoroacetoacetate 1 and thiourea
R2
O
R1
HO
F3C
NH
4
3
1
2 N
S
H
4a-q
Fig. 1. RSR configuration with lowest conformational energy of À41.18 kJ molÀ1
.
3 in refluxing THF for 24 h.14 The hexahydropyrimidine 4a–q
(Table 1), considered to be an intermediate in the Biginelli reaction,

O. C. Agbaje et al. / Bioorg. Med. Chem. Lett. 21 (2011) 989–992
991
Br
CF3
O
F3C
OEt
CF3 OEt
F3C OH
HN
O
F3C
HN
OEt
p-TsOH
6
O
HN
O
O
O
toluene, reflux
S
N
H
pyridine,EtOH,
reflux, 3h
86%
O
O
O
O
S
N
H
56%
N
H
S
O
7
4a
5
IC50 = 8.1 µM
IC50 = 12.2 µM
Scheme 1. Synthesis of fluorinated tetrahydropyrimidine derivatives 5 and 7.14
was isolated in moderate to excellent yields (67–95%).14 Hexahy-
dropyrimidine 4 contains three stereogenic carbon centers, which
may exist as four possible diastereomers. However spectral exam-
ination (1H NMR) of products 4a–q, confirmed the formation of
one isomer only.14
Acknowledgment
We are thankful to the U.S. Department of Education Title III
Grant, Tennessee State University, for financial support.
The above observation was confirmed by characteristic signals
for two doublets, which correspond to the two trans-axial methane
protons (H3–H4) in 4 (Fig. 1). The observed coupling constants
(J = 11.1–11.8 Hz) are in agreement with the values previously
reported.11,14 On a theoretical point of view, we determined the
conformational energies of all possible diastereomers using Mae-
stro Software version 8.0 Schrodinger, Inc. The large conformational
energies of the phenyl and alkoxycarbonyl (acyl) groups favor fixed
conformations where these groups take an equatorial orientation,
with the RSR configuration having the lowest conformational
energy (Fig. 1).
The IR spectra of products 4a–q, showed sharp and intense sig-
nals for the hydroxyl functionality in all cases.
We examined the antiproliferative activity of the fluorinated
hexahydropyrimidine (4a–q) derivatives on human colon (COLO
320 HSR) cancer cell line in the 72 h drug exposure Alamarblue™
assays (Table 1).15
References and notes
1. Filler, R.; Banks, R. E. Organofluorine Chemicals and Their Industrial Applications;
Ellis Horwood: Chichester, UK, 1979.
2. [a] Welch, J. T. Tetrahedron 1987, 43, 3123; b Frezza, M.; Balestrino, D.; Soulere,
L.; Reverchon, S.; Queneau, Y.; Forestier, C.; Doutheau, A. Eur. J. Org. Chem. 2006,
4731; c Buscemi, S.; Pace, A.; Piccionello, A.; Macaluso, G.; Vivona, N. J. Org.
Chem. 2005, 70, 3288 (and references cited therein).
3. [a] Schlosser, M. In Enantiocontrolled Synthesis of Fluoroorganic Compounds:
Stereochemical Challenges and Biomedical Targets; Soloshonok, V. A., Ed.; Wiley:
Chichester, 1999; p 613; b Michel, D.; Schlosser, M. Tetrahedron 2000, 56, 4253.
4. Park, B. K.; Kitterringham, N. R.; O’Neill, P. M. Annu. Rev. Pharmacol. 2001, 41,
443.
5. [a] Sing, R. P.; Shreeve, J. M. Tetrahedron 2000, 56, 7613; b Lin, P.; Jiang, J.
Tetrahedron 2000, 56, 3635; c Andrew, R. J.; Mellor, J. M.; Reid, G. Tetrahedron
2000, 56, 7255; d Andrew, R. J.; Mellor, J. M.; Reid, G. Tetrahedron 2000, 56,
7261; e Andrew, R. J.; Mellor, J. M.; Reid, G. Tetrahedron 2000, 56, 7267; f
Tyvorskii, V. I.; Bobrov, D. N.; Kulinkovich, O. G.; Aelterman, W.; De Kimpe, N.
Tetrahedron 2000, 56, 7313.
6. [a] Prakash, G. K. S.; Yudin, A. K. Chem. Rev. 1997, 999, 757–786; b Mandal, M. J.
Fluorine Chem. 2001, 112, 123; c Singh, R. P.; Shreeve, J. M. Tetrahedron 2000, 56,
7613.
7. Bohacek, R. S.; McMartin, C.; Guida, C. W. Med. Res. Rev. 1996, 16, 3.
8. [a] Russowski, D.; Canto, R. F. S.; Sanches, S. A. A.; D’Oca, M. G. M.; Fatima, A. D.;
Carvalho, J. E. D. Bioorg. Chem. 2006, 34, 173; b Kappe, C. O. Tetrahedron 1993,
49, 6937; c Kappe, C. O. Eur. J. Med. Chem. 2000, 35, 1043; d Tu, S.; Miao, C.;
Fang, F.; Youjian, F.; Li, T.; Zhuang, Q.; Zhang, X.; Zhu, S.; Shi, D. Bioorg. Med.
Chem. Lett. 2004, 14, 1533.
9. [a] Mayer, T. U.; Kapoor, T. M.; Haggarty, S. J.; King, R. W.; Schreiber, S. L.;
Mitchison, T. J. Science 1999, 289, 971; b Gartner, M.; Sunder-Plassmann, N.;
Seiler, J.; Utz, M.; Vernos, I.; Surrey, T.; Giannis, A. ChemBioChem 2005, 6, 1173;
c Sarli, V.; Giannis, A. Clin. Cancer Res. 2008, 14, 7583.
10. [a] Braekman, J. C.; Daloze, D.; Flammang, R.; Maquestiau, A. Org. Mass
Spectrom. 1989, 24, 837; b Drandarov, K.; Guggisberg, A.; Hesse, M. Helv. Chim.
Acta 1999, 82, 229.
The dose of the compound that inhibited 50% cell proliferation
(IC50) was calculated using the data generated from the assay. It
was found that among the series of fluorinated hexahydropyrimi-
dine derivatives 4e, 4o, 4p and 4q (entries 5, 15, 16 and 17) exhibited
noactivityatIC50 greaterthan100
4i, 4j, 4m and 4n showed only moderate activity with IC50 value
ranging from 51–75 M (Table 1, entries 1–4, 8–10 and 13–14).
lM. Compounds4a, 4b, 4c, 4d, 4h,
l
Among the 17 fluorinated Hexahydropyrimidine derivatives
4a–q, only two derivatives 4g and 4k containing naphthyl-substi-
tuted analogs (R = naphthyl, Table 1, entries 7 and 11) showed the
highest activity with IC50 values of 9.3 and 9.9
(Table 1), also compound 4f showed good activity of IC50 value of
11. 5 M (Table 1, entry 6).
lM, respectively
11. Saloutin, V. I.; Burgart, Ya. V.; Kuzueva, O. G.; Kappe, C. O.; Chupakhin, O. N. J.
Fluorine Chem. 2000, 103, 17.
12. Kappe, C. O.; Falson, S. F. Synlett 1998, 718.
13. Lakshmikantham, M. V.; Mitchell, K. J. J. Org. Chem. 1969, 34, 2665.
14. Experimental and spectral data for title compounds.
l
Upon refluxing compound 4a in toluene in the presence of
p-toluenesulfonic acid (PTSA), fluorinated tetrahydropyrimidine 5
was formed via azeotropic removal of water 5.14 To further opti-
mize the synthesis of tetrahydropyrimidine, equal ratios of 5 with
bromo trifluoromethyl phenyl ethanone 6 was refluxed in EtOH/
pyridine to give fluorinated sulfanyl tetrahydropyrimidine 7 in
good yield (Scheme 1).14 Both compounds showed good antiprolif-
erative activity against colon cancer cell line COLO 320 with IC50
General procedure for the PPE-mediated preparation of Hexahydropyrimidine
4:
A
mixture of the appropriate aldehyde
2
(2.0 mmol), ethyl
trifluoroacetoacetate 1 (2.0 mmol), thiourea 3 (3.0 mmol), and THF (10 ml)
containing 300 mg PPE was heated under reflux for the time indicated in
Table 1. After cooling, the reaction mixture was poured onto 10 g of crushed
ice. Stirring was continued for several hours; the solid products were filtered,
washed with ice water and subsequently dried to give 4.
Furan-2-yl(4-hydroxy-6-(4-methoxyphenyl)-2-thioxo-4-(trifluoromethyl) hexahy-
dropyrimidin-5-yl)methanone 4f: white solid, mp: 207 °C; IR (nujol) 3398, 3180,
2217, 1675, 1209 cmÀ1 1H NMR (CD3OD, 400 MHz) d 3.70 (s, 3H), 4.67 (d,
.
values of 12.2 and 8.1 lM, respectively (Scheme 1).
J = 11.6 Hz, 1H), 5.03 (d, J = 11.6 Hz, 1H), 6.52 (m, 1H), 6.80 (m, 2H), 7.26 (m,
3H), 7.69 (s, 1H); EIMS m/z: 137 (20%), 166 (100%), 167 (15%), 205 (15%), 207
(30%) (M + 1).
In conclusion, the preliminary anticancer studies showed that 4f,
4g, 4k, 5 and 7 represent novel leads for further development. Com-
pound 7 was the most active among the series and will be subjected
to in-depth structure–activity relationship (SAR). Also further bio-
logical evaluation of the fluorinated hexahydropyrimidine, tetrahy-
dropyrimidine and other new derivatives against mitotic motor Eg5
(kinesin spindle protein, KSP) is underway, and results will be re-
ported in due course.
(6-(4-Fluorophenyl)-4-hydroxy-2-thioxo-4-(trifluoromethyl) hexahydropyrimidin-
5-yl)(naphthalen-2-yl)methanone 4g: brown solid, mp: 190–192 °C; 3397, 3210,
2219, 1675, 1210 cmÀ1 1H NMR (CD3OD, 400 MHz) d 4.61 (d, J = 11.6 Hz, 1H), 5.00
.
(d, J = 11.6 Hz, 1H), 7.29 (m, 4H), 7.57 (m, 2H), 7.84 (m, 3H), 8.01 (m, 1H), 8.33 (s,
1H); EIMS m/z: 137 (20%), 166 (100%), 167 (15%), 205 (15%), 207 (30%) (M + 1).
(6-(Benzo[d][1,3]dioxol-5-yl)-4-hydroxy-2-thioxo-4-(trifluoromethyl)hexahydropy-
rimidin-5-yl)(naphthalen-2-yl)methanone 4k: yellowsolid, mp:207–209 °C;3398,

992
O. C. Agbaje et al. / Bioorg. Med. Chem. Lett. 21 (2011) 989–992
1H NMR (CD3OD, 400 MHz) d 4.60 (d, J = 11.6 Hz, in 86% yield as yellow solid. Mp: 215 °C; IR (nujol) 3295, 3180, 1710, 1670 cmÀ1
.
3210, 2217, 1670, 1210 cmÀ1
.
1H), 5.01 (d, J = 11.6 Hz, 1H), 6.0 (s, 2H), 6.57 (d, J = 8.0 Hz, 1H), 6.87 (m, 1H), 7.03
(s, 1H), 7.58(m, 2H), 7.84 (m, 3H), 8.0 (d, J = 8.0 Hz, 1H), 8.35(s, 1H);EIMSm/z:137
(20%), 166 (100%), 167 (15%), 205 (15%), 207 (30%) (M + 1).
1H NMR (CD3OD, 400MHz) d 1.09 (t, 3H), 3.58(s, 2H), 4.06 (q, 2H), 5.6 (s, 1H), 6.0
(s, 2H) 6.4–6.88 (m, 3H), 7.6 (d, 2H), 7.87(d, 2H). LC/MS: 563 (M + 1).
15. Materials and methods: Human colon (COLO 320 HSR) cancer cell line was
obtained from the American Type Culture Collection (Manassas, VA). Cell line
was maintained in RPMI 1640 supplemented with 10% fetal bovine serum
(FBS), 0.15% sodium bicarbonate, 0.011% sodium pyruvate, 0.24% Hepes and
10 ml/L of antibiotic solution. The cells were grown in 150 cm2 culture plates
in an air/CO2 (95:5) atmosphere at 37 °C and passaged approximately every 3
days. Cancer cell line (1 Â 104 cells per well) was plated using RPMI 1640
medium containing FBS in 96-well plates and left to attach for 24 h. Cells were
then treated with either vehicle (DMSO) or the indicated concentrations of
different compounds. Cells were exposed for a total of 72 h to test compounds
and vehicle controls. Alamarblue™ reagent dye was added to wells in a 1:10
following exposure period and incubated at 37 °C overnight. The plates were
then analyzed using a microtiter plate reader at dual wavelengths (560 nm k
excitation, 590 nm k emission). Each experiment was done in triplicate and
results are expressed as means for each determination. The IC50 values were
calculated using linear regression method and are expressed in micromolar
Ethyl-4-(benzo[d][1,3]dioxol-5-yl)-2-thioxo-6-(trifluoromethyl)-1,2,3,4-tetrahydropy-
rimidine-5-carboxylate 5: (2.0 mmol) in 15 ml of toluene, 0.1 g of p-
toluenesulfonic acid was added. The mixture was refluxed for 6 h with
azeotropic removal of water. The reaction mixture was cooled to room
temperature and filtered from
a
small amount of solid material.
Recrystallization of this residue from ethanol/water gave 5 in 56% yield as
white solid. Mp: 147 °C; IR (nujol) 3295, 3180, 2217, 1705, 1675, 1560, 1209,
1130 cmÀ1.1HNMR(CD3OD, 400 MHz) d 1.09 (t, 3H), 4.06(q,2H), 5.4 (s, 1H), 6.0 (s,
2H), 6.77 (d, 2H), 6.9 (s, 1H); EIMS m/z; 326 (5%), 375 (100%) (M + 1).
Ethyl-4-(benzo[d][1,3]dioxol-5-yl)-2-(2-oxo-2-(4-(trifluoromethyl)phenyl) ethylthio)-
6-(trifluoromethyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate 7:
A mixture of
2-bromo-1-[4-(trifluoromethyl)phenyl]ethanone 6 (1 mmol) and ethyl-4-(1,3-
benzodioxol-5-yl)-2-thioxo-6-(trifluoromethyl)-1,2,3,4-tetrahydropyrimidine-5-
carboxylate
5
(1 mmol) was refluxed in ethanol (5 ml) containing
pyridine(1 mmol) for 3 h. The reaction mixture was cooled to room
temperature. The solid obtained was filtered, dried. Further purification by
flash column chromatography on silica using hexane/ethyl acetate (1:1) gave 7
(lM).