Synthesis of fluoromethyl-containing analogs of antitumor alkaloid luotonin A

Article abstract of DOI:10.1007/s11172-010-0064-9

A convenient method for the synthesis of hitherto unknown 3-bromomethyl-2-chloro-4-fluoromethylquinolines has been developed. Coupling of 3-bromomethyl-2-chloro-4-trifluoromethylquinoline with 4(3H)-quinazolinone with subsequent intramolecular Heck cyclization leads to 7-trifluoromethylluotonin, an analog of the antitumor alkaloid luotonin A. 7-Trifluoromethylluotonin retains the antitumor activity including apoptosis of cultured tumor cells and inhibiting DNA-topoisomerase I.

Full text of DOI:10.1007/s11172-010-0064-9

Russian Chemical Bulletin, International Edition, Vol. 59, No. 1, pp. 209—218, January, 2010
209
Synthesis of fluoromethylꢀcontaining analogs
of antitumor alkaloid luotonin A
A. S. Golubev,a V. O. Bogomolov,a A. F. Shidlovskii,a L. G. Dezhenkova,b
A. S. Peregudov,a A. A. Shtil,c and N. D. Chkanikova
aA. N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences,
28 ul. Vavilova, 119991 Moscow, Russian Federation.
Fax: +7 (499) 135 5085. Eꢀmail: golubev@ineos.ac.ru
bG. F. Gause Research Institute of New Antibiotics, Russian Academy of Medical Sciences,
11 ul. B. Pirogovskaya, 119021 Moscow, Russian Federation
cN. N. Blokhin Russian Cancer Scientific Center, Russian Academy of Medical Sciences,
24 Kashirskoe shosse, 115478 Moscow, Russian Federation
A convenient method for the synthesis of hitherto unknown 3ꢀbromomethylꢀ2ꢀchloroꢀ4ꢀ
fluoromethylquinolines has been developed. Coupling of 3ꢀbromomethylꢀ2ꢀchloroꢀ4ꢀtrifluoꢀ
romethylquinoline with 4(3H)ꢀquinazolinone with subsequent intramolecular Heck cyclization
leads to 7ꢀtrifluoromethylluotonin, an analog of the antitumor alkaloid luotonin A. 7ꢀTrifluoꢀ
romethylluotonin retains the antitumor activity including apoptosis of cultured tumor cells and
inhibiting DNAꢀtopoisomerase I.
Key words: camptothecin, luotonin A, antitumor alkaloid, topoisomerase I, 4ꢀfluoromethꢀ
ylꢀ2ꢀquinolones, intramolecular Heck cyclization.
Camptothecin (1) is a pentacyclic alkaloid isolated
from the tree Camptotheca acuminata.1 Camptothecin reꢀ
mains one of the most promising compounds for developꢀ
ment of antitumor drugs.2 In the 1980s, it was found that
camptothecin is an efficient inhibitor of the DNAꢀdepenꢀ
dent enzyme, topoisomerase I in the eukaryotic cells.3 Acꢀ
cording to modern concepts, the inhibitory activity of
camptothecin is due to the formation of a threeꢀcompoꢀ
nent complex camptothecin—topoisomerase I—DNA,4
which causes singleꢀstrand cleavage of DNA. To date, nuꢀ
merous waterꢀsoluble camptothecin analogs have been
synthesized; two of them, topotecan (hycampin) and irinoꢀ
tecan (camptosar), are used in clinical practice and about
dozen of others undergo clinical trial.5
cally labile sixꢀmembered αꢀhydroxylactone ring,9 as well
as introduction of lipophilic groups into the ring B of campꢀ
tothecin.10 It is important to study naturally occurring
alkaloid luotonin A (2), the structural analog of camptothꢀ
ecin,11 since its derivatives are suitable for the search for
new camptothecinꢀbased antitumor drugs.
Despite this progress, clinical application of antitumor
drugs based on camptothecin faces a number of problems.6
The major problem is the lability of the lactone ring E at
physiological рH values, with the openꢀchain form of the
lactone ring (the carboxyl form) possessing only 10% acꢀ
tivity of the lactone form. Another problem of the campꢀ
tothecin derivatives is their high general resorption toxiciꢀ
ty. Chemotherapy causes emergence of multiple drug reꢀ
sistance of tumors because of poor accumulation of the
drugs due to their active transport from cells.7
Several approaches have been suggested to increase the
camptothecin stability, in particular, replacement of the
lactone ring by an aromatic one,8 introduction of a sevenꢀ
membered βꢀhydroxylactone ring instead of the metaboliꢀ
One of the most convenient approaches to the syntheꢀ
sis of luotonin A is formation of the ring C of the pentacyꢀ
cle by fusion of the A/Bꢀ and D/Eꢀfragments through the
Nꢀalkylation of 4(3H)ꢀquinazolinone with 3ꢀbromomethꢀ
ylꢀ2ꢀhaloquinolines and subsequent intramolecular Heck
cyclization (Scheme 1).12 Thus, the substituted 3ꢀbroꢀ
momethylꢀ2ꢀhaloquinolines are key building blocks in the
synthesis of modified luotonins.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 205—214, January, 2010.
1066ꢀ5285/10/5901ꢀ0209 © 2010 Springer Science+Business Media, Inc.

210
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Golubev et al.
Scheme 1
Scheme 2
X = Br, Cl
The purpose of this work is the synthesis and assessꢀ
ment of antitumor activity of luotonin A derivatives conꢀ
taining fluoromethyl substituents in the ring B of the penꢀ
tacycle.13 Our approach to the synthesis of fluoromethyl
analogs of luotonin A is based on the use of new building
blocks, viz., 3ꢀbromomethylꢀ2ꢀchloroꢀ4ꢀfluoromethylꢀ
quinolines 3 (Scheme 2). In this work, we describe a methꢀ
od for the synthesis of quinolines 3 containing 4ꢀCF3ꢀ,
4ꢀCF2Hꢀ, and 4ꢀCF2Cl groups. The synthesis of 3ꢀbroꢀ
momethylꢀ2ꢀchloroꢀ6ꢀfluoroꢀ4ꢀtrifluoromethylquinoline
illustrates a possibility of introduction of a substituent
(R´ = F, see Scheme 2) into the benzene ring of quinoline
3 in addition to the 4ꢀCF3ꢀgroup. This convenient method
for the synthesis of quinolines 3 opens a direct route (see
Scheme 1) to luotonins containing different fluoromethyl
groups in position 7* of the pentacycle essential for the
physiological activity: a lipophilic group (CF3); a group
capable of forming hydrogen bonds (CF2H); a group that
can be alkylated with endogenous nitrogen bases (CF2Cl).
More important is that these building blocks can be used
for the synthesis of 7ꢀfluoromethylꢀsubstituted camptothꢀ
ecins, homocamptothecins, 14ꢀazocamptothecins, and
other compounds of the camptothecin series.
R = H, Br; R´ = H, F; X = H, F, Cl; Pg = COCMe3, COCH2Me
by coupling at the keto group, and, finally, the lactam ring
closure. In the case of the synthesis of 4ꢀtrifluoromethylꢀ
2ꢀquinolones, this scheme is advantageous as compared to
the classic Knorr synthesis of 2ꢀquinolones. In the frameꢀ
work of the method developed,15 3ꢀmethylꢀ4ꢀtrifluoroꢀ
methylꢀ2ꢀquinolone 4a (Х = F, R´ = H, see Scheme 2)
was synthesized starting from NꢀВocꢀaniline using pyroꢀ
phoric ButLi as a lithiating agent.
In the synthesis of quinolines 3 based on the Schlosser
approach,15 we modified the stage of orthoꢀfluoroacetylaꢀ
tion of anilines. We chose a pivaloyl group (Pg = COCMe3,
R = H) and a propionyl group (Pg = COCH2Me, R = Br,
see Scheme 2) as protecting and orthoꢀdirecting groups.
Safe BuLi was a lithiating agent and ethyl trifluoroacetate,
ethyl difluoroacetate, and ethyl chlorodifluoroacetate were
used for the acylation.
Lithiation of Nꢀpivaloylaniline (5a) and Nꢀpivaloylꢀpꢀ
fluoroaniline (5b) was performed in THF with a threefold
excess of BuLi in the temperature range from –10 to
0 °C for 3 h (see Ref. 16). The acylating agent, ethyl trifluꢀ
oroacetate (2 equiv.), was added at –70 °C and, after stanꢀ
dard workꢀup, products 6a and 6b were obtained in preꢀ
parative yields (~50%) (Scheme 3). However, in the case
of ethyl difluoroacetate and ethyl chlorodifluoroacetate,
the yields of the corresponding ketones were unsatisfactory.
Ketones 6c and 6d were successfully obtained starting
from Nꢀpropionylꢀ2ꢀbromoaniline 5c (Scheme 4). They
were isolated in >50% yields. Lithiation with BuLi was
performed in this case at –75 °C in THF.17 It should be
noted that the lithiation of 2ꢀbromoaniline 5c is accompaꢀ
nied by formation of Nꢀpropionylꢀ2ꢀnꢀbutylaniline 5d
To preparate quinolines 3, we developed a fiveꢀstep
synthetic scheme. NꢀProtected anilines served as the startꢀ
ing compounds, synthesis of quinolones 4 being a key step
(see Scheme 2).
A scheme for the assembly of 4ꢀtrifluoromethylꢀsubstiꢀ
tuted 2ꢀquinolone core has been described earlier.15 This
includes orthoꢀtrifluoroacetylation of aniline bearing an
appropriate Nꢀprotecting group, introduction of a C2ꢀfragment
* Two general ways for the luotonin A numbering were used. The
first is based on the camptothecin numeration, see Refs 12c and
14a. The second uses the IUPAC nomenclature, see Refs 13b,c,
and 14b.

Fluoroanalog of antitumor alkaloid luotonin A
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
211
Scheme 3
Scheme 5
R = H (5a, 6a), F (5b, 6b)
Reactants and conditions: 1) BunLi, –10—0 °C; 2) CF3CO2Et, –75 °C.
Scheme 4
R
R´
H
F
H
H
X
F
F
H
Cl
7a
7b
7c
7d
CMe3
CMe3
CH2Me
CH2Me
Conditions: toluene, 100 °C.
Scheme 6
X = H (6c), Cl (6d)
Reactants and conditions: 1) BunLi, –75 °C; 2) CF2XCO2Et, –75 °C.
(see Ref. 18) the yield of which reached 10%. The lithiaꢀ
tion of 2ꢀbromoaniline 5c results also in the formation
of noticeable amounts (up to 10%) of debromination
product, Nꢀpropionylaniline 5e.
R´
H
F
H
H
X
F
F
H
Cl
4a
4b
4c
4d
The formation of the carbon skeleton of 4ꢀfluoromethꢀ
ylꢀ2ꢀquinolones involved introduction of the C2ꢀfragment
by coupling ketones 6a—d with 1ꢀ(ethoxycarbonylꢀ
ethylidene)triphenylphosphorane15 (Scheme 5). The Witꢀ
tig reaction products 7a—d were isolated by column chroꢀ
matography as mixtures of E,Zꢀisomers, which were charꢀ
Reactants and conditions: 6 M HCl or 2 M HCl.
reaction requires short time and proceeds in high yields.
Upon bromination with NBS in boiling CCl4 in the presꢀ
ence of a radical initiator,19 quinolines 8a—d give the tarꢀ
get quinolines 3a—d (see Scheme 7).
1
acterized by H and 19F NMR spectra and used in the
subsequent steps of the lactam ring formation as such
(Scheme 6). Compounds 7a,b underwent cyclization to
form 2ꢀquinolones 4a,b upon refluxing in a mixture of 6M
HCl and acetic acid. However, the crucial factor for the
preparation of 2ꢀquinolones 4c,d was performing the cyꢀ
clization of compounds 7c,d under less drastic conditions,
viz., upon refluxing in 2M HCl.
Quinoline 3a was successfully used in the synthesis of
7ꢀtrifluoromethylluotonin A. The reaction of 3a with
4(3H)ꢀquinazolinone in DMF in the presence of potassiꢀ
um tertꢀbutoxide20 occurs at 0 °C to form compound 9
(Scheme 8). Subsequent intramolecular Heck cyclization
in the presence of Pd(OAc)2, tri(cyclohexyl)phosphine,
and potassium acetate in DMF at 160 °C (see Ref. 20)
afforded 7ꢀtrifluoromethylluotonin 10 in 30% yield
(Scheme 8).
2ꢀQuinolones 4a—d upon refluxing in an excess of
POCl3 furnish 2ꢀchloroquinolines 8a—d (Scheme 7). The

212
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Golubev et al.
Scheme 7
Table 1. Yields and some physicochemical characteristics of comꢀ
pounds 3a—d, 4b—d, 5d, 6a—d, 8a—d, 9, 10
Comꢀ Yield
M.p.
/°C
Found
Calculated
Molecular
formula
(%)
poꢀ
und
(%)
С
Н
N
3a
3b
3с
3d
4b
4c
4d
5d
6а
6b
6с
6d
8а
8b
8с
8d
9
74
72
37
47
60—61
(LP)
95—96
(LP)
67—68
(LP)
65—66
(LP)
40.87 1.86 4.24 C11H6BrClF3N
40.71 1.86 4.32
38.59 1.54 4.05 C11H5BrClF4N
38.57 1.47 4.09
42.95 2.15 4.34 C11H7BrClF2N
43.10 2.30 4.57
38.91 1.75 4.07 C11H6BrCl2F2N
38.75 1.77 4.11
65 213—214 53.76 2.79 5.61 C11H7F4NO
(ethanol) 53.89 2.88 5.71
30 229—230 62.91 4.30 6.78 C11H9F2NO
(ethanol) 63.16 4.34 6.70
32 215—216 54.11 3.35 5.82 C11H8ClF2NO
(ethanol) 54.23 3.31 5.75
—
47
48
60
51
91
91
R´
H
F
H
H
X
F
F
H
Cl
3a, 4a, 8a
3b, 4b, 8b
3c, 4c, 8c
3d, 4d, 8d
79—80
(LP)
yellow
oil
47—48
(LP)
94—95
76.07 9.41 6.65 C13H19NO
76.06 9.33 6.82
57.04 5.11 4.98 C13H14F3NO2
57.14 5.16 5.13
53.49 4.48 4.80 C13H13F4NO2
53.61 4.50 4.81
58.09 4.90 6.14 C11H11F2NO2
Scheme 8
(EA—LP) 58.15 4.88 6.16
yellow
oil
57—58
(LP)
64—65
(LP)
50.51 3.94 5.29 C11H10ClF2NO2
50.49 3.85 5.35
53.94 2.91 5.69 C11H7ClF3N
53.79 2.87 5.70
50.09 2.21 5.11 C11H6ClF4N
50.12 2.29 5.31
72 119—120 58.09 3.60 6.17 C11H8ClF2N
(LP)
66—67
(LP)
58.04 3.54 6.15
50.32 2.67 5.24 C11H7Cl2F2N
50.41 2.69 5.34
78
60 190—191 58.61 2.75 10.82 C19H11ClF3N3O
(EA—LP) 58.55 2.84 10.78
30 256—258 64.61 2.79 11.78 C19H10F3N3O
(СН2Cl2— 64.59 2.85 11.89
LP)
10
Structures of all the compounds obtained were conꢀ
Activity of topoisomerase I. Compound 10 caused inhiꢀ
firmed by elemental analysis data (Table 1) and their specꢀ
tral characteristics (Table 2).
bition of topoisomerase I activity at relatively high conꢀ
centration, 40 μmol L–1 (Fig. 1). Even at this concentraꢀ
tion, only insignificant amount of the rapidly migrating
plasmid was found. The lower concentrations had no efꢀ
fect of retarding untwisting of DNA. Like for cytotoxicity,
the range of concentrations inhibiting topoisomerase I
demonstrates low activity of 10 with respect to topoꢀ
isomerase I. Hence, a conclusion can be drawn that the
luotonin derivative synthesized suppresses the topoiꢀ
somerase I activity only in relatively high concentrations.
Thus, we developed an efficient and reliable scheme for
the synthesis of hitherto unknown 3ꢀbromomethylꢀ2ꢀchloꢀ
Biological
Cytotoxicity. Compound 10 caused apoptosis of culꢀ
tured human colon adenocarcinoma and leukemia cells in
relatively high concentrations: 50% growth inhibitory conꢀ
centrations after cell exposure for 72 h were 17 3 and
21 4 μmol L–1, respectively. These values are lower than
those found for the most antitumor compounds (microꢀ
molar and submicromolar ranges).

Fluoroanalog of antitumor alkaloid luotonin A
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
213
Table 2. Spectral characteristics of compounds 3a—d, 4b—d, 5d, 6a—d, 8a—d, 9, 10
Comꢀ
pound
NMR (CDCl3), δ (J/Hz)
MS (EI, 70 eV),
m/z (Irel (%))
1Н
13С
19F
3а
3b
4.96 (s, 2 H, CH2Br);
25.55 (q, JC,F = 6.1), 123.07, 123.72
(q, JC,F = 278.3), 125.00 (q, JC,F = 5.1),
128.42, 128.96, 129.49, 131.56,
24.73 (s, CF3)
327 [M + Н]+ (4),
325 [M + Н]+ (11),
245 (39), 244 (17),
243 (100), 209 (17),
208 (31), 204 (75),
176 (44), 140 (14)
345 [M + Н]+ (18),
343 [M + Н]+ (31),
263 (40), 262 (17),
261 (100), 226 (26),
225 (40), 207 (59),
176 (57), 158 (44)
7.76 (t, 1 H, Ar, J = 7.8);
7.89 (t, 1 H, Ar, J = 7.5);
8.15 (d, 1 H, Ar, J = 8.5);
8.28 (dm, 1 H, Ar, J = 8.5)
134.81 (q, JC,F = 30.2), 147.51, 151.65
5.01 (s, 2 H, CH2Br);
7.67 (td, 1 H, Ar, J = 7.7,
J = 2.5); 7.91 (dm, 1 H, Ar, (d, JC,F = 26.2), 123.51 (q, JC,F = 278.7),
J = 9.7); 8.15 (dd, 1 H, Ar,
J = 9.3, J = 5.5)
25.28 (q, JC,F = 5.5), 109.45
(qd, JC,F = 26.2, JC,F = 5.2), 121.99
–31.54 (s, F, 1 F);
22.02 (s, CF3, 3 F)
124.00 (d, JC,F = 10.6), 129.52,
132.00 (d, JC,F = 9.5), 134.08
(qd, JC,F = 31.0, JC,F = 5.8), 144.63,
151.01, 161.63 (d, JC,F = 251.7)
24.50, 112.10 (t, JC,F = 241.3), 123.41,
124.68 (t, JC,F = 3.4), 128.18
3c
4.95 (s, 2 H, CH2Br);
7.47 (t, 1 H, CF2H,
J = 53.0); 7.74 (td, 1 H,
Ar, J = 7.4, J = 1.4);
–32.66 (s, СF2Н)
309 [M + Н]+ (15),
307 [M + Н]+ (20),
228 (40), 226 (100),
190 (32), 140 (45)
(t, JC,F = 5.2), 128.53, 129.41, 131.57,
138.31 (t, JC,F = 20.7), 147.72, 150.59
7.88 (td, 1 H, Ar, J = 7.4,
J = 0.9); 8.14 (dd, 1 H, Ar,
J = 8.4, J = 0.9); 8.36 (dm,
1 H, Ar, J = 8.4)
3d
5.00 (s, 2 H, CH2Br);
7.75 (td, 1 H, Ar, J = 7.1,
J = 1.4); 7.88 (td, 1 H, Ar,
J = 8.4, J = 1.1); 8.14 (dd,
1 H, Ar, J = 8.4, J = 0.9);
8.36 (dm, 1 H, Ar, J = 8.4)
2.32 (q, 3 H, Me, J = 3.7);
7.24 (t, 1 H, Ar, J = 7.6);
7.38 (d, 1 H, Ar, J = 8.3);
7.53 (t, 1 H, Ar, J = 7.6);
7.70 (d, 1 H, Ar, J = 7.1);
12.33 (br.s, 1 Н, NH)
2.38 (q, 3 H, Me, J = 3.9);
7.36—7.42 (m, 2 H, Ar);
7.54 (td, 1 H, Ar, J = 8.5,
J = 2.4); 12.39 (br.s,
26.20 (t, JC,F = 8.2), 122.07, 125.07
(t, JC,F = 300.1), 125.30 (t, JC,F = 7.2), system,
126.44, 128.70, 129.57, 131.48,
140.21 (t, JC,F = 23.5), 147.67, 151.84
34.34 (d, АВ
343 [M + Н]+ (19),
341 [M + Н]+ (31),
305 (13), 261 (60),
259 (83), 224 (85),
190 (100), 189 (89),
140 (75)
228 [M + Н]+ (30),
227 [M]+ (100),
199 (82), 198 (40),
158 (27), 130 (93)
JАВ = 169.7),
35.43 (d, АВ
system,
JАВ = 169.7)
23.60 (s, CF3)
4a*
4b*
13.27 (q, JC,F = 4.4), 114.66, 116.34,
122.97, 124.62 (q, JC,F = 278.6),
124.67 (q, JC,F = 4.4), 130.63,
131.85 (q, JC,F = 28.8), 132.82,
137.82, 161.31
13.43 (q, JC,F = 4.4), 109.96
–40.89 (s, F, 1 F),
23.60 (s, CF3, 3 F)
246 [M + Н]+ (30),
245 [M]+ (100),
217 (43), 216 (86),
207 (22), 176 (22),
148 (80)
(qd, JC,F = 26.5, JC,F = 5.5), 115.12
(d, JC,F = 7.7), 118.18 (d, JC,F = 8.9),
118.90 (d, JC,F = 24.3), 124.35
(q, JC,F = 278.6), 131.30 (qd,
1 Н, NH)
JC,F = 29.9, JC,F = 3.3), 134.54, 134.64,
157.53 (d, JC,F = 237.7), 160.96
12.21, 114.38 (t, JC,F = 237), 115.42,
116.18, 122.52, 125.00, 130.22,
132.09 (t, JC,F = 8), 135.56 (t,
JC,F = 22), 138.03, 161.70
4c*
2.28 (t, 3 H, Me, J = 1.8);
7.23 (dd, 1 H, Ar, J = 7.7,
J = 1.0); 7.37 (d, 1 H, Ar,
J = 7.7); 7.51 (t, 1 H, Ar,
J = 7.7); 7.58 (t, 1 H, CF2H,
J = 52.7); 7.93 (d, 1 H, Ar,
J = 8.2); 12.13 (br.s,
–35.37 (s, СF2Н)
210 [M + Н]+ (30),
209 [M]+ (100),
181 (22), 180 (36),
162 (17), 161 (20),
130 (53)
1 Н, NH)
4d**
2.57 (t, 3 H, Me, J = 4.8);
7.35 (dd, 1 H, Ar, J = 7.6,
J = 8.2); 7.41 (d, 1 H, Ar,
J = 8.2); 7.59 (dd, 1 H, Ar,
J = 8.2, J = 7.6); 8.06
14.00 (t, JC,F = 6.9), 114.09, 116.49,
122.81, 125.09 (t, JC,F = 6.6),
126.07 (t, JC,F = 293.7), 130.57, 130.60,
137.41 (t, JC,F = 23.3), 137.99, 161.38
33.98 (s, СF2Сl)
245 [M]+ (8),
243 [M]+ (21),
209 (18), 208 (100),
207 (12), 180 (76),
179 (23), 130 (14)
(d, 1 H, Ar, J = 8.2);
11.40 (br.s, 1 Н, NH)
(to be continued)

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Golubev et al.
Table 2 (continued)
Comꢀ
pound
NMR (CDCl3), δ (J/Hz)
MS (EI, 70 eV),
m/z (Irel (%))
1Н
13С
19F
5d
1.00 (t, 3 H, (CH2)3Me,
J = 7.3); 1.34 (t, 3 H,
9.96, 13.98, 22.60, 30.55, 31.13, 32.00,
124.23, 125.37, 126.59, 129.47, 134.16,
135.11, 172.31
—
205 [M]+ (11),
176 (72), 148 (17),
120 (14), 107 (18),
106 (100)
CH2Me, J = 7.3), 1.44 (m,
2 H, (CH2)2CH2Me); 1.63
(m, 2 H, CH2CH2CH2Me);
2.49 (q, 2 H, CH2Me,
J = 7.3); 2.63 (t, 2 H,
CH2(CH2)2Me, J = 7.3); 7.07
(br.s, 1 H, NH); 7.13—7.29
(m, 3 H, Ar); 7.87 (d, 1 H, Ar,
J = 7.5)
6а
1.35 (s, 9 H, 3 Me);
27.51, 40.61, 115.35, 116.62
8.49 (s, CF3)
273 [M]+ (4),
7.17 (ddd, 1 H, Ar, J = 8.3,
J = 7.3, J = 1.1); 7.68 (ddd, 131.85 (q, JC,F = 4.4), 137.76, 144.06,
(q, JC,F = 291.2), 121.21, 122.43,
230 (9), 205 (15),
204 (100), 207 (24),
189 (14), 146 (15),
121 (7), 120 (56)
1 H, Ar, J = 8.7, J = 7.3,
J = 1.6); 7.96 (dm, 1 H, Ar,
J = 8.3); 8.88 (dd, 1 H, Ar,
J = 8.7, J = 1.0); 11.24
(br.s, 1 Н, NH)
178.38, 182.70 (q, JC,F = 33.7)
6b
6с
1.42 (s, 9 H, 3 Me);
27.48, 40.55, 115.85 (d, JC,F = 6.3),
116.31 (q, JC,F = 291.4); 117.28
(d.q, JC,F = 24.4, JC,F = 4.3); 123.25
(d, JC,F = 6.9), 125.24 (d, JC,F = 21.8);
140.51, 156.71 (d, JC,F = 244.8);
178.26, 182.30 (q, JC,F = 35.1)
–40.01 (s, F, 1 F);
8.00 (s, CF3, 3 F)
292 [M + Н]+ (7),
291 [M]+ (13),
248 (12), 223 (14),
222 (100), 207 (24),
164 (19), 139 (19),
138 (52)
7.51 (ddd, 1 H, Ar, J = 9.4,
J = 7.3, J = 3.0); 7.70 (dm,
1 H, Ar, J = 9.4); 8.98 (dd,
1 H, Ar, J = 9.4, J = 5.0);
11.14 (br.s, 1 Н, NH)
1.35 (t, 3 H, Me, J = 7.5);
2.56 (q, 2 H, CH2, J = 7.5); 116.77, 121.03, 122.40, 131.36
6.45 (t, 1 H, CF2H, J = 53.4); (t, JC,F = 4.3); 137.06, 142.95, 173.25,
7.22 (td, 1 H, Ar, J = 8.2,
J = 0.8); 7.73 (td, 1 H, Ar,
J = 8.6, J = 1.2); 8.04 (dd,
1 H, Ar, J = 8.2,J = 1.5);
8.90 (d, 1 H, Ar, J = 8.6);
11.22 (br.s, 1 Н, NH)
9.41, 31.70, 110.52 (t, JC,F = 253.4);
–43.03 (s, CF2Н)
18.87 (s, CF2Сl)
24.71 (s, CF3)
227 [M]+ (5),
177 (14), 176 (100),
171 (15), 149 (14),
120 (95)
190.10 (t, JC,F = 24.1)
6d
1.34 (t, 3 H, Me, J = 7.5);
2.56 (q, 2 H, CH2, J = 7.5); (t, JC,F = 304.9); 121.40, 122.26, 132.00
9.41, 31.70, 114.46, 119.85
263 [M]+ (13),
261 [M]+ (42),
206 (75), 176 (43),
121 (33), 120 (100)
7.21 (ddd, 1 H, Ar, J = 8.5,
J = 7.3, J = 1.3); 7.72 (ddd,
1 H, Ar, J = 8.5, J = 7.3,
J = 1.3); 8.15 (dm, 1 H, Ar,
J = 8.5, J = 1.0); 8.89 (dd,
1 H, Ar, J = 8.5, J = 1.0);
11.00 (br.s, 1 Н, NH)
2.82 (q, 3 H, Me, J = 2.8);
7.70 (dd, 1 H, Ar, J = 8.2,
J = 7.3); 7.81 (dd, 1 H, Ar,
J = 8.2, J = 7.3); 8.11
(d,1 H, Ar, J = 8.6); 8.26
(d,1 H, Ar, J = 8.6)
2.76 (q, 3 H, Me, J = 2.9);
7.54 (td, 1 H, Ar, J = 8.3,
J = 2.4); 7.84 (d, 1 H, Ar,
(t, JC,F = 4.9); 137.41, 143.74, 173.16,
183.85 (t, JC,F = 28.4)
8а
8b
17.85 (q, JC,F = 4.6); 123.27, 124.18
(q, JC,F = 5.1); 124.28 (q, JC,F = 278.2);
128.40, 129.25, 129.87 (q, JC,F = 2.0);
130.11, 134.20 (q, JC,F = 30.2); 146.37,
152.94
247 [M]+ (30),
245 [M]+ (100),
210 (13), 209 (25),
190 (35), 189 (73),
140 (20)
17.88 (q, JC,F = 4.4); 108.68 (qd,
JC,F = 26.5, JC,F = 5.5); 120.38
(d, JC,F = 25.4); 124.07 (d, JC,F = 10.0);
–30.93 (s, F, 1 F);
23.99 (s, CF3, 3 F)
265 [M]+ (30),
263 [M]+ (60),
228 (42), 209 (43),
208 (100), 158 (81)
J = 11.0); 8.05 (dd, 1 H, Ar, 124.09 (q, JC,F = 278.6); 131.05, 131.75
J = 9.2, J = 5.7)
(d, JC,F = 10.0); 133.71 (qd, JC,F = 30.5,
JC,F = 6.6); 143.54, 152.32 (d, JC,F = 3.3);
161.40 (d, JC,F = 249.9)
(to be continued)

Fluoroanalog of antitumor alkaloid luotonin A
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
215
Table 2 (continued)
Comꢀ
pound
NMR (CDCl3), δ (J/Hz)
MS (EI, 70 eV),
m/z (Irel (%))
1Н
13С
19F
8с
2.76 (t, 3 H, Me, J = 1.6);
7.42 (t, 1 H, CF2H,
J = 53.5); 7.66 (ddd, 1 H,
Ar, J = 8.4, J = 7.3, J = 1.1); (t, JC,F = 22.1); 146.52, 152.38
7.80 (dd, 1 H, Ar, J = 8.4,
16.08, 112.79 (t, JC,F = 230.1); 123.67,
123.72 (t, JC,F = 1.9); 127.97, 129.11
(t, JC,F = 5.5); 129.22, 130.04, 136.89
–33.31 (s, СF2Н)
229 [M]+ (37),
227 [M]+ (100),
192 (5), 172 (12),
171 (28)
J = 7.3); 8.11 (d, 1 H, Ar,
J = 8.4); 8.31 (dd, 1 H, Ar,
J = 8.4, J = 1.1)
8d
2.79 (t, 3 H, Me, J = 4.0);
7.70 (ddd, 1 H, Ar, J = 8.6,
J = 7.0, J = 1.4); 7.80 (ddd,
1 H, Ar, J = 8.4, J = 7.0,
J = 1.0); 8.11 (dd, 1 H, Ar,
J = 8.4, J = 1.0); 8.31 (dm,
1 H, Ar, J = 8.6)
18.42 (t, JC,F = 6.6); 122.26, 124.45
(t, JC,F = 7.7); 125.59 (t, JC,F = 294.1);
128.11, 128.16, 129.31, 130.00, 139.68
(t, JC,F = 24.3); 146.54, 152.98
35.56 (s, СF2Cl)
263 [M]+ (20),
261 [M]+ (34),
228 (32), 227 (12),
226 (100), 191 (10),
190 (33), 188 (20)
9
5.65 (q, 2 H, CH2, J = 1.0); 45.44 (q, JC,F = 4.4); 121.78, 122.93,
7.51 (ddd, 1 H, Ar, J = 8.2, 123.63 (q, JC,F = 278.6); 124.63,
J = 7.0, J = 1.4); 7.67—7.80 124.94 (q, JC,F = 5.5); 126.75, 127.55,
24.80 (s, CF3)
355 [M + Н – Сl]+ (22),
341 [M – Сl]+ (100),
227 (11), 226 (59)
(m, 3 H, Ar); 7.85 (s, 1 H,
H(2));7.87 (ddd, 1 H, Ar,
127.55, 129.24, 129.54, 132.00, 134.57,
137.60 (q, JC,F = 29.8); 144.57, 147.63,
J = 8.2, J = 7.0, J = 1.4); 8.12 147.99, 151.82, 161.04
(dd, 1 H, Ar, J = 8.4, J = 0.9);
8.20—8.31 (m, 2 H, Ar)
5.62 (q, 2 H, CH2, J = 2.4); 47.93 (q, JC,F = 5.5); 121.22, 123.60,
10
20.73 (s, СF3)
354 [M + Н]+ (9),
353 [M]+ (42),
325 (13), 306 (23),
292 (26), 291 (38),
277 (39), 235 (26),
219 (26), 203 (31),
189 (71), 147 (100)
7.68 (t, 1 H, Ar, J = 7.0);
7.89—7.98 (m, 2 Н, Ar);
8.03 (dd, 1 H, Ar, J = 7.8,
J = 7.1); 8.20 (d, 1 H, Ar,
J = 8.1); 8.37 (d, 1 H, Ar,
J = 8.1); 8.52 (d, 1 H, Ar,
J = 7.8); 8.66 (d, 1 H,
Ar, J = 8.6)
123.74 (q, JC,F = 278.6); 124.07
(q, JC,F = 2.2), 126.40, 127.16
(q, JC,F = 2.2), 127.76, 128.70, 130.20
(q,JC,F = 33.2), 130.26, 131.27, 131.52,
134.70, 148.92, 150.06, 151.11, 151.32,
160.04
* 1H, 13C, and 19F NMR spectra were recorded in DMSOꢀd6.
** 13C NMR spectrum was obtained in DMSOꢀd6.
roꢀ4ꢀfluoromethylquinolines 3, which are promising buildꢀ
ing blocks for the synthesis of 7ꢀfluoromethylꢀcontaining
alkaloids of the camptothecin series, in particular, 7ꢀtriꢀ
fluoromethylluotonin 10. It was found that 7ꢀtrifluoromeꢀ
thylluotonin 10 retains some antitumor activity, causing
death of tumor cells and inhibiting topoisomerase I. Using
the method developed for the synthesis of 7ꢀfluoromethylꢀ
substituted luotonins, we hope to optimize the structure of
topoisomerase I inhibitors of the camptothecin series, the
prototypes of the targetꢀspecific antitumor drugs.
Experimental
1
H and 13C{1H} NMR spectra were recorded on a Bruker
AvanceTM600 spectrometer (600.22 MHz and 150.925 MHz,
respectively). Chemical shifts for protons and 13C nuclei were
determined with respect to the residual signal for chloroform
(7.27 ppm) or the signal for CDCl3 (77.0 ppm), respectively and
recalculated from the SiMe4 signal. Chemical shifts were deterꢀ
mined with accuracy no less than 0.001 ppm and 0.03 ppm,
respectively. 19F {1H} NMR spectra were recorded on a Bruꢀ
ker AvanceTM300 spectrometer (288.38 MHz). Chemical
shifts for 19F nuclei were determined with respect to trifluoroꢀ
DNAst Topo I 0.1
1
5
10
20
40
C/μmol L–1
Fig. 1. Influence of compound 10 on the activity of topoisomerasꢀ
es I in vitro. C is concentration of compound 10. DNAst is
a supertwisted DNA.

216
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Golubev et al.
acetic acid as an external standard with accuracy no less
than 0.01 ppm.
lated first as light yellow crystals. Further elution of the column
gave first Nꢀ(2ꢀnꢀbutylphenyl)propionamide (5d) (0.32 g) and
then Nꢀphenylpropionamide (5e) (0.2 g), m.p. 103—104 °C (from
LP); see Ref. 24: 103—104 °C. 1H NMR spectrum of compound
5e completely agreed with that reported in the literature.24
Nꢀ[2ꢀ(Chlorodifluoroacetyl)phenyl]propionamide (6d) was
obtained from Nꢀ(2ꢀbromophenyl)propionamide (5c) and ethyl
chlorodifluoroacetate similarly the synthesis of compound 6c as
a yellowish dense liquid.
Mass spectra were obtained on a Finnigan Polaris Q instruꢀ
ment (ion trap, 70 eV, the DIP procedure was used for the samꢀ
ple injection) and on a MSꢀ30 Kratos instrument (70 eV, temperꢀ
ature of ionizing chamber was 200 °C, a system for direct injecꢀ
tion of the sample was used). Silica gel with the particle size of
0.06—0.20 mm (Merck Kieselgel 60) was used for column chroꢀ
matography. Monitoring of the reaction course and purity of
products obtained was performed using Merck Kieselgel 60 F254
TLC plates. nꢀButyllithium (1.6 M solution in hexane) was purꢀ
chased from Acros. Tetrahydrofuran (Aldrich) was prepared for
the lithiation reaction by reflux over sodium metal in the presꢀ
ence of benzophenone until dark blue color of the solution was
persistent with subsequent distillation under argon. All the lithiaꢀ
tion reactions were performed under argon. Elemental analysis
was performed in the Laboratory of Elemental Analysis of the A.
N. Nesmeyanov Institute of Organoelement Compounds, Rusꢀ
sian Academy of Sciences. Light petroleum (LP) and ethyl aceꢀ
tate (EA) were used as the solvents.
Ethyl 4,4,4ꢀtrifluoroꢀ3ꢀ[2ꢀ(2,2ꢀdimethylpropionylamino)ꢀ
phenyl]ꢀ2ꢀmethylbutꢀ2ꢀenoate (7a) (a mixture of E,Zꢀisomers in
the ratio 3 : 1). A solution of (1ꢀethoxycarbonylethylidene)ꢀ
triphenylphosphorane (0.77 g, 2.1 mmol) and compound 6a (0.58 g,
2.1 mmol) in anhydrous toluene (10 mL) was heated for 6 h at
100 °C. Toluene was evaporated in vacuo. The residue was exꢀ
tracted with boiling pentane (3×30 mL). A precipitate formed
after cooling pentane was filtered off. The pentane extracts were
concentrated. The residue was purified by column chromatograꢀ
phy on silica gel using a LP—EA (6 : 1) solvent mixture as an
eluent to obtain compound 7a (0.55 g) as a colorless dense liquid.
1
NꢀSubstituted anilines 5a,b were synthesized using a general
procedure.21 Physicochemical and spectral characteristics of
Nꢀphenylꢀ2,2ꢀdimethylpropionamide22 (5a) and Nꢀ(4ꢀfluoꢀ
rophenyl)ꢀ2,2ꢀdimethylpropionamide16b (5b) completely agreed
with the data reported in the literature. Nꢀ(2ꢀBromophenyl)ꢀ
propionamide (5c) was synthesized using procedure given in
work.23
The yield was 73%. H NMR of the predominant isomer in the
mixture of isomers (CDCl3), δ: 0.91 (t, 3 H, CH2Me, J = 7.1 Hz);
1.35 (s, 9 H, 3 Me); 2.36 (q, 3 H, Me(2), J = 2.2 Hz); 3.96 (q, 2 H,
CH2Me, J = 7.1 Hz); 7.11—7.14 (m, 2 H, Ar); 7.43 (td, 1 H, Ar,
J = 8.4 Hz, J = 4.5 Hz); 7.60 (br.s, 1 H, NH); 8.16 (d, 1 H, Ar,
J = 8.2 Hz). 19F NMR of the predominant isomer in the mixture
1
of isomers (CDCl3), δ: 18.96 (s, CF3). H NMR of the minor
Compounds 3a—d, 4a—d, 5d, 5e, 8a—d are colorless crystalꢀ
line substances.
isomer in the mixture of isomers (CDCl3), δ: 1.35 (s, 9 H, 3 Me);
1.42 (t, 3 H, CH2Me, J = 7.1 Hz); 1.83 (q, 3 H, Me(2), J = 2.4 Hz);
4.39 (q, 2 H, CH2Me, J = 7.14 Hz); 7.18—7.24 (m, 2 H, Ar);
7.51 (td, 1 H, Ar, J = 8.4 Hz, J = 4.5 Hz); 7.52 (br.s, 1 H, NH);
8.36 (d, 1 H, Ar, J = 8.2 Hz). 19F NMR of the minor isomer in
the mixture of isomers (CDCl3), δ: 15.61 (s, CF3). No analyticalꢀ
ly pure sample was obtained.
Nꢀ[2ꢀ(Trifluoroacetyl)phenyl]ꢀ2,2ꢀdimethylpropionamide (6a).
nꢀButyllithium (1.6 M solution in hexane, 78.2 mL, 125.1 mmol)
was added over 1 h to a solution of Nꢀphenylꢀ2,2ꢀdimethylpropiꢀ
onamide (5a) (7.4 g, 41.7 mmol) in anhydrous THF (80 mL) at
temperatures from –10 °C to 0 °C. A suspension obtained was
stirred for 3 h at –5—0 °C. Then the reaction mixture was cooled
to –70 °C, followed by addition of ethyl trifluoroacetate (20.9 g,
147.1 mmol). The reaction mixture was stirred for 1 h at –70 °C,
slowly warmed to 0 °C, treated with saturated aqueous NH4Cl
(50 mL) and ethyl acetate (150 mL), the water layer was separated.
The organic layer was washed with brine (2×50 mL), dried with
MgSO4, and concentrated. The residue was separated by column
chromatography eluting with a LP—EA (6 : 1) solvent mixture to
obtain compound 6a (5.4 g) as a yellowish dense liquid.
Nꢀ[4ꢀFluoroꢀ2ꢀ(trifluoroacetyl)phenyl]ꢀ2,2ꢀdimethylpropiꢀ
onamide (6b) was obtained similarly from Nꢀ(4ꢀfluorophenyl)ꢀ
2,2ꢀdimethylpropionamide (5b) and ethyl trifluoroacetate. The
product as light yellow crystals was isolated by column chromaꢀ
tography on silica gel with LP—CH2Cl2 (1 : 1) solvent mixture as
the eluent.
Nꢀ[2ꢀ(Difluoroacetyl)phenyl]propionamide (6c). nꢀButyllithꢀ
ium (1.6 M solution in hexane, 10.5 mL, 33 mmol) was added to
a solution of Nꢀ(2ꢀbromophenyl)propionamide (5c) (3.64 g,
16 mmol) in anhydrous THF (50 mL) at –75 °C. The solution
obtained was stirred for 1 h at –75 °C followed by addition of
ethyl difluoroacetate (3.97 g, 32 mmol). The reaction mixture
was stirred for 1 h at –75 °C, slowly warmed to 0 °C, treated with
saturated aq. NH4Cl (10 mL) and ethyl acetate (50 mL), the
water layer was separated. The organic layer was washed with
brine (2×20 mL), dried with MgSO4, and concentrated. The
residue was separated by column chromatography eluting with
a LP—EA (3 : 1) solvent mixture. Compound 6c (2.2 g) was isoꢀ
Ethyl 3ꢀ[5ꢀfluorophenylꢀ2ꢀ(2,2ꢀdimethylpropionylamino)]ꢀ
4,4,4ꢀtrifluoroꢀ2ꢀmethylbutꢀ2ꢀenoate (7b) (a mixture of E,Zꢀisoꢀ
mers in the ratio 4 : 1) was obtained from compound 6b similarly
to the preparation and isolation of compound 7a. Colorless dense
liquid. The yield was 70%. 1H NMR of the predominant isomer
in the mixture of isomers (CDCl3), δ: 0.97 (t, 3 H, CH2Me,
J = 7.0 Hz); 1.33 (s, 9 H, 3 Me); 2.35 (q, 3 H, Me(2), J = 2.0 Hz);
4.01 (q, 2 H, CH2Me, J = 7.0 Hz); 6.89 (dd, 1 H, Ar, J = 8.3 Hz,
J = 2.8 Hz); 7.13 (td, 1 H, Ar, J = 8.4 Hz, J = 2.5 Hz); 7.53 (br.s,
1 H, NH); 8.05 (dd, 1 H, Ar, J = 8.4 Hz, J = 5.3 Hz). 19F NMR
of the predominant isomer in the mixture of isomers (CDCl3), δ:
–40.01 (s, 1 F); 19.05 (s, 3 F). 1H NMR of the minor isomer in
the mixture of isomers (CDCl3), δ: 1.34 (s, 9 H, 3 Me); 1.41 (t, 3 H,
CH2Me, J = 7.0 Hz); 1.83 (q, 3 H, Me(2), J = 2.0 Hz); 4.39 (q, 2 H,
CH2Me, J = 7.3 Hz); 6.99 (dd, 1 H, Ar, J = 8.3 Hz, J = 3.0 Hz);
7.20 (td, 1 H, Ar, J = 8.4 Hz, J = 2.7 Hz); 7.42 (br.s, 1 H, NH);
8.27 (dd, 1 H, Ar, J = 8.4 Hz, J = 5.0 Hz). 19F NMR of the minor
isomer in the mixture of isomers (CDCl3), δ: –39.34 (s, 1 F);
15.61 (s, 3 F). No analytically pure sample was obtained.
Ethyl 4,4ꢀdifluoroꢀ2ꢀmethylꢀ3ꢀ(2ꢀpropionylaminophenyl)butꢀ
2ꢀenoate (7c) (a mixture of E,Zꢀisomers in the ratio 1 : 2) was
obtained from compound 6c similarly to the synthesis of comꢀ
pound 7a. A predominant isomer with higher Rf was isolated by
column chromatography on silica gel as colorless crystals eluting
with a LP—EA (2 : 1) solvent mixture. M.p. 68—69 °C. 1H NMR
of the predominant isomer (CDCl3), δ: 1.27 (t, 3 H, CH2Me,
J = 7.5 Hz); 1.43 (t, 3 H, OCH2Me, J = 7.2 Hz); 1.79 (t, 3 H,

Fluoroanalog of antitumor alkaloid luotonin A
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
217
Me(2), J = 3.4 Hz); 2.42 (q, 2 H, CH2Me, J = 7.5 Hz); 4.40 (q, 2 H,
OCH2Me, J = 7.2 Hz); 7.07 (t, 1 H, CF2H, J = 55.0 Hz);
7.07—7.27 (m, 3 H, Ar, NH); 7.49 (t, 1 H, Ar, J = 7.0 Hz); 8.22
(d, 1 H, Ar, J = 7.0 Hz). 19F NMR of the predominant isomer
(CDCl3), δ: –36.99 (d, AВꢀsystem, JAВ = 319.1 Hz); –34.09
(d, AВꢀsystem, JAВ = 319.1 Hz). 13C NMR of the predominant
isomer (CDCl3), δ: 9.64, 14.08, 17.35, 31.92, 61.98, 111.91
(t, JC,F = 234.5 Hz), 122.75, 123.64, 124.51, 129.61, 129.90,
135.54, 135.74 (t, JC,F = 24.4 Hz), 138.48 (t, JC,F = 8.3 Hz),
166.97, 172.04. MS (EI, 70 eV), m/z (Irel (%)): 311 [M]+ (6), 255
(19), 254 (23), 210 (59), 182 (89), 181 (26), 162 (100). Found (%):
C, 61.79; H, 6.27; N, 4.44. C16H19F2NO3. Calculated (%):
C, 61.73; H, 6.15; N, 4.50. The minor isomer was isolated by
further elution in a mixture with the predominant isomer.
1H NMR of the minor isomer in the mixture of isomers (CDCl3),
δ: 0.88 (t, 3 H, OCH2Me, J = 7.0 Hz); 1.27 (t, 3 H, CH2Me,
J = 7.5 Hz); 2.29 (t, 3 H, Me(2), J = 1.0 Hz); 2.43 (q, 2 H,
CH2Me, J = 7.5 Hz); 3.94 (q, 2 H, OCH2Me, J = 7.0 Hz); 6.59
(t, 1 H, CF2H, J = 56.0 Hz); 7.07—7.27 (m, 3 H, Ar, NH); 7.40
(t, 1 H, Ar, J = 7.0 Hz); 8.11 (d, 1 H, Ar, J = 7.3 Hz). 19F NMR
of the minor isomer in the mixture of isomers (CDCl3), δ: –37.13
17.6 mmol) was refluxed for 1 h. The reaction mixture was cooled.
Unreacted phosphorus oxychloride was evaporated in vacuo
(12 Torr). The residue was poured in ice. Crystals formed were
filtered off, washed with saturated aq. NaHCO3 on the filter to
pH 7, dried in air to obtain compound 8a (0.78 g).
2ꢀChloroꢀ6ꢀfluoroꢀ3ꢀmethylꢀ4ꢀtrifluoromethylquinoline (8b),
2ꢀchloroꢀ4ꢀdifluoromethylꢀ3ꢀmethylquinoline (8c), and 2ꢀchloroꢀ
4ꢀchlorodifluoromethylꢀ3ꢀmethylquinoline (8d) were obtained
similarly from the corresponding 3ꢀmethylquinolinꢀ2(1H)ꢀones.
3ꢀBromomethylꢀ2ꢀchloroꢀ4ꢀtrifluoromethylquinoline (3a).
A solution of 8a (0.25 g, 1 mmol), Nꢀbromosuccinimide (0.2 g,
1.1 mmol), and 1,1´ꢀazobis(cycleоhexanecarbonitrile) (10 mg)
in dry CCl4 (10 mL) was refluxed for 24 h. The reaction course
was monitored by TLC using a LP—EA solvent mixture (25 : 1)
as an eluent, Rf of the starting compound was 0.4; Rf of the
product was 0.3. The solvent was evaporated, the residue was
purified by column chromatography on silica gel with a mixture
of LP and EA (15 : 1) as an eluent to obtain compound 3a (0.24 g).
3ꢀBromomethylꢀ2ꢀchloroꢀ6ꢀfluoroꢀ4ꢀtrifluoromethylquinoline
(3b), 3ꢀbromomethylꢀ2ꢀchloroꢀ4ꢀdifluoromethylquinoline (3c) and
3ꢀbromomethylꢀ2ꢀchloroꢀ4ꢀchlorodifluoromethylquinoline (3d)
were obtained similarly from corresponding 2ꢀchloroꢀ3ꢀmethꢀ
ylquinolines.
(d, AВꢀsystem, JAВ = 313.6 Hz); –34.65 (d, AВꢀsystem, JAВ
=
= 313.6 Hz). The overall yield of two isomers was 63%.
Ethyl 4ꢀchloroꢀ4,4ꢀdifluoroꢀ2ꢀmethylꢀ3ꢀ(2ꢀpropionylaminoꢀ
phenyl)butꢀ2ꢀenoate (7d) (a mixture of E,Zꢀisomers in the ratio
2 : 3) was obtained from compound 6d similarly to the preparaꢀ
tion and isolation of compound 7a. Yellowish dense liquid. The
yield was 53%. 1H NMR of the predominant isomer (CDCl3), δ:
1.32 (t, 3 H, CH2Me, J = 7.5 Hz); 1.44 (t, 3 H, OCH2Me,
J = 7.2 Hz); 1.79 (t, 3 H, Me(2), J = 3.4 Hz); 2.47 (q, 2 H,
CH2Me, J = 7.5 Hz); 4.40 (q, 2 H, OCH2Me, J = 7.2 Hz);
7.20—7.37 (m, 3 H, Ar, NH); 7.51 (t, 1 H, Ar, J = 7.5 Hz); 8.31
(d, 1 H, Ar, J = 8.2 Hz). 19F NMR of the predominant isomer
(CDCl3), δ: 27.83 (d, AВꢀsystem, JAВ = 161.3 Hz); 29.33 (d, AВꢀsysꢀ
tem, JAВ = 161.3 Hz). 1H NMR of the minor isomer in a mixture
of isomers (CDCl3), δ: 0.92 (t, 3 H, OCH2Me, J = 7.0 Hz);
1.27 (t, 3 H, CH2Me, J = 7.5 Hz); 2.39 (t, 3 H, Me(2), J = 1.0 Hz);
2.43 (q, 2 H, CH2Me, J = 7.5 Hz); 3.94 (q, 2 H, OCH2Me,
J = 7.0 Hz); 7.20—7.37 (m, 3 H, Ar, NH); 7.44 (t, 1 H, Ar,
J = 7.0 Hz); 8.13 (d, 1 H, Ar, J = 8.2 Hz). 19F NMR of the minor
isomer in a mixture of isomers (CDCl3), δ: 31.55 (d, AВꢀsystem,
JAВ = 161.3 Hz); 32.98 (d, AВꢀsystem, JAВ = 161.3 Hz). No anaꢀ
lytically pure sample was obtained.
3ꢀMethylꢀ4ꢀtrifluoromethylquinolinꢀ2(1H)ꢀone (4a). Comꢀ
pound 7a (0.55 g, 1.5 mmol) was added to a mixture of HCl (6 M,
5 mL) and acetic acid (5 mL) and this was refluxed for 6 h
Crystals formed after cooling were filtered off and washed with
water on the filter until the washings were neutral to obtain comꢀ
pound 4a (0.22 g, 62%). M.p. 235—236 °C (from ethanol);
Ref. 15: 237—239 °C.
6ꢀFluoroꢀ3ꢀmethylꢀ4ꢀtrifluoromethylquinolinꢀ2(1H)ꢀone (4b)
was obtained from compound 7b similarly to compound 4a.
4ꢀDifluoromethylꢀ3ꢀmethylquinolinꢀ2(1H)ꢀone (4c). Comꢀ
pound 7c (2.23 g, 7.2 mmol) was refluxed for 4 h in HCl (2 M,
40 mL). Crystals formed after cooling were filtered off and washed
with water on the filter until the washings were neutral to obtain
compound 4c (0.63 g).
3ꢀ[(2ꢀChloroꢀ4ꢀtrifluoromethylquinolinꢀ3ꢀyl)methyl]quinaꢀ
zolinꢀ4(3H)ꢀone (9). Potassium tertꢀbutoxide (0.2 g, 1.8 mmol)
was added to a solution of 4(3H)ꢀquinazolinone (0.24 g, 1.65 mmol)
in dry DMF (20 mL). The reaction mixture was stirred for 1 h at
20 °C and cooled to 0 °C, followed by a dropwise addition of a
solution of 3a (0.52 g, 1.6 mmol) in DMF (10 mL). The reaction
mixture was stirred for 1 h at 0 °C, warmed to 20 °C, diluted
with 5% aq. Na2CO3 to pH 10, and extracted with ethyl acetate
(3×30 mL). The organic extracts were combined, washed with
brine, dried with MgSO4, and concentrated. The product was
purified by column chromatography on silica gel eluting with
a LP—EA (1 : 1) solvent mixture to obtain compound 10 (350 mg)
as grayish crystals.
7ꢀTrifluoromethylluotonin or 14ꢀtrifluoromethylquino[2´,3´:3,4]ꢀ
pyrrolo[2,1ꢀb]quinazolinꢀ11(13H)ꢀone (10). Compound 9 (0.27
g, 0.69 mmol), Pd(OAc)2 (16 mg, 0.07 mmol), tri(cyclohexyl)ꢀ
phosphine (40 mg, 0.14 mmol), and potassium acetate (137 mg,
1.4 mmol) were mixed in a flask equipped with an inlet for an
inert gas. Dry DMF (15 mL) was added to the mixture, a suspenꢀ
sion obtained was stirred for 30 min under argon at 20 °C, then
warmed to 160 °C, and kept for 15 min at this temperature. The
reaction mixture was warmed to 20 °C, diluted with 5% aq.
Na2CO3 to pH 10, and extracted with ethyl acetate (3×30 mL).
The organic extract were combined, washed with brine, dried
with MgSO4, and concentrated. The product was purified by
column chromatography on silica gel eluting with a LP—EA
(1 : 1) solvent mixture to obtain compound 10 (73 mg) as grayish
crystals.
Biological testing. Cytotoxicity. The method25 is based on the
reduction of tetrazolium salt to formazan in living cells. Comꢀ
pound 10 was dissolved in dimethyl sulfoxide to final concentraꢀ
tion of 10 mmol L–1. This solution was used for the preparation
of the drug dilutions in the medium. The К562 leukemia and
HCТ116 colon carcinoma cell lines were propagated in Dulbecꢀ
co modified Eagle's medium supplemented with 5% fetal calf
serum, Lꢀglutamine (2 mmol L–1), penicillin (100 unit mL–1),
streptomycin (100 μg mL–1)). Cells were plated on 96ꢀwell plates
(3•103 cells in 190 μL of cultural medium). Compound 10 was
4ꢀChlorodifluoromethylꢀ3ꢀmethylquinolinꢀ2(1H)ꢀone (4d) was
obtained from compound 7d similarly to compound 4c.
2ꢀChloroꢀ3ꢀmethylꢀ4ꢀtrifluoromethylquinoline (8a). A soluꢀ
tion of 4a (0.8 g, 3.5 mmol) in phosphorus oxychloride (2.7 g,

218
Russ.Chem.Bull., Int.Ed., Vol. 59, No. 1, January, 2010
Golubev et al.
added at final concentrations 0.2—50 μmol L–1. Control wells
were filled with the medium. Each concentration of 10 was testꢀ
ed in triplicates. The cells were incubated for 72 h at 37 °C, 5% of
CO2, then aqueous solution of tetrazolium bromide (2.5 μg mL)
was added. After 2 h, formazan was dissolved in dimethyl sulfoxꢀ
ide (100 μL), and optical density was measured at 540 nm on an
LKB spectrophotometer (Sweden). The percentage of dying cells
at given concentration of 10 was calculated as average optical
density in the wells with the agent divided by average optical
density of control wells.
Modulation of topoisomerase I activity. The method is based
on the comparison of electrophoretic mobility of DNA dependꢀ
ing on its conformation, i.e., degree of relaxation under the acꢀ
tion of topoisomerase I. Inhibition of the enzyme prevents the
relaxation of the supercoiled plasmid DNA, therefore, the unꢀ
wound DNA migrates faster in the gel compared to relaxed DNA.
The influence of compound 10 on topoisomerase I activity was
studied as described.26 The reaction mixture (20 μL) containꢀ
ing DNA (0.25 μg) (pBR322 plasmid; Fermentas, Lithuania) and
compound 10 was incubated for 30 min at 37 °C in the buffer:
TRISꢀHCl (35 mmol L–1, pH 8.0), KCl (72 mmol L–1), MgCl2
(5 mmol L–1), dithiothreitol (5 mmol L–1), spermidine (2 mmol L–1),
bovine serum albumin (100 μg mL–1); all the reagents were from
Sigma, USA. The reaction was terminated by addition of sodium
dodecyl sulfate (final concentration 1%), proteinase K was addꢀ
ed for 40 min at 37 °C. The samples were analyzed by electroꢀ
phoresis in 1% agarose gel (3 V cm–1) for 4—5 h in the buffer
cotaining TRISꢀbase (40 mmol L–1), EDTA (1 mmol L–1), glaꢀ
cial acetic acid (30 mmol L–1). The gels were stained with ethiꢀ
dium bromide (0.5 μg mL–1) and visualized under the UV light.
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This work was financially supported by the Russian Founꢀ
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Received October 8, 2009