2-Cyanoaminopyrimidines as a class of antitumor agents that promote tubulin polymerization

Article abstract of DOI:10.1016/j.bmcl.2007.03.070

A series of 4-chloro-2-cyanoamino-6-fluoroalkylamino-5-phenylpyrimidines was prepared as a result of our efforts to modify a series of [1,2,4]triazolo[1,5-a]pyrimidines that proved to be potent anticancer agents with a unique mechanism of tubulin inhibiti

Full text of DOI:10.1016/j.bmcl.2007.03.070

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3003–3005
2-Cyanoaminopyrimidines as a class of antitumor agents
that promote tubulin polymerization
Nan Zhang,a,* Semiramis Ayral-Kaloustian,a Thai Nguyen,a
Richard Hernandezb and Carl Beyerb
aDiscovery Medicinal Chemistry, Chemical and Screening Sciences, Wyeth Research,
401 North Middletown Road, Pearl River, NY 10965, USA
bOncology Research, Wyeth Research, 401 North Middletown Road, Pearl River, NY 10965, USA
Received 31 January 2007; revised 21 March 2007; accepted 21 March 2007
Available online 25 March 2007
Abstract—A series of 4-chloro-2-cyanoamino-6-fluoroalkylamino-5-phenylpyrimidines was prepared as a result of our efforts to
modify a series of [1,2,4]triazolo[1,5-a]pyrimidines that proved to be potent anticancer agents with a unique mechanism of tubulin
inhibition. On the cyanoamino nitrogen, a methyl group is optimal for activity among alkyl groups introduced. The structure–activ-
ity relationship for the rest of the molecule resembles that of [1,2,4]triazolo[1,5-a]pyrimidines. A lead compound (5) retained in vitro
potency compared with TTI-237. In the mechanism of action studies, it behaved in the same manner as TTI-237. In addition, it is
also capable of overcoming multidrug resistance due to P-gp. These findings strongly suggest that this series of 2-cyanoaminopyr-
imidines binds at the same site and in the same binding mode as TTI-237. Further modifications of the 2-cyanoamino group are
underway.
Ó 2007 Elsevier Ltd. All rights reserved.
CF3
Antimitotic agents that disrupt microtubule dynamics
have attracted much interest in development of antican-
cer therapies.1 We recently reported a series of
[1,2,4]triazolo[1,5-a]pyrimidines as small, synthetic anti-
cancer agents with a unique mechanism of action in pro-
moting tubulin polymerization.2 This series of
compounds promotes tubulin polymerization in vitro,
but does not bind competitively with paclitaxel. The lead
compound of this series, TTI-237, is currently in Phase I
clinical trials. As a subsequent effort to modify the
[1,2,4]triazolo[1,5-a]pyrimidine core, we developed a
new series of compounds, 2-cyanoaminopyrimidines,
that retained in vitro potency and the unique mechanism
of action observed with [1,2,4]triazolo[1,5-a]pyrimidines.
We now report the synthesis, SAR, and mechanism of
action studies for this series of compounds (Schemes 1
and 2).
H
F
O
N
CH3
N
NH
N
F
TTI-237
N
N
Cl
Starting from 5-chloro-6-(2,4,6-trifluorophenyl)-N-(2,2,
2-trifluoroethyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine
1,2 treatment with 1 equivalent of sodium hydride in the
presence of an excess amount of iodomethane in DMSO
led to an isomeric mixture of mono-methylated com-
pounds.3 These isomers were separated and their struc-
tures were elucidated with extensive NMR studies.
One of the methylated compounds, N-[5-chloro-3-
methyl-6-(2,4,6-trifluorophenyl)[1,2,4]triazolo[1,5-a]pyr-
imidin-7(3H)-ylidene]-2,2,2-trifluoroethanamine (2), was
further treated with 1 equiv sodium hydride in DMSO. It
underwent a ring-opening reaction to afford 4-chloro-6-
[(2,2,2-trifluoroethyl)amino]-5-(2,4,6-trifluorophenyl)pyr-
imidin-2-yl(methyl)cyanamide (3a).4 Compounds 3b and
3c were prepared analogously, using iodoethane and allyl
bromide instead of iodomethane.
Keywords: 2-Cyanoaminopyrimidines; Antitumor agents; Tubulin
polymerization.
*
As in the case of [1,2,4]triazolo[1,5-a]pyrimidines,2 the
4-fluoro group can be replaced in the presence of the
Corresponding author. Tel.: +1 845 602 3425; fax: +1 845 602
5561; e-mail: zhangn@wyeth.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.070

3004
N. Zhang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3003–3005
F
F
F
F
F
F
CF3
N
N
N
CF3
NH
CF3
N
NH
CH3I/NaH
rt, 18 h
NaH
N
N
N
F
rt, 1 h
F
CH3
N
CH3
F
Cl
N
N
Cl
N
N
Cl
CN
3a
1
2
Scheme 1. Synthesis of 2-cyanoaminopyrimidines.
F
F
F
O
CF3
N
NH
N
(CH2)nN(CH3)2
CF3
N
NH
HO(CH2)nN(CH3)2, NaH
DMSO, 60 oC, 4h
N
N
F
CH3
F
CH3
Cl
N
Cl
CN
CN
3a
Scheme 2. Replacement of the 4-fluoro group.
4
2- and 6-fluoro groups. Thus, treatment of compound 3a
with amino alcohols, in the presence of sodium hydride in
DMSO, afforded compound 4 exclusively. Compound 5
was prepared analogously, using (1S)-2,2,2-trifluoro-1-
methylethylamine2 instead of 2,2,2-trifluoroethylamine.
the interaction as in 4a. However, with a shorter two-
methylene tether, the dimethylamino group in 4b has a
much harder time to form the same interaction. Replac-
ing the achiral 2,2,2-trifluoroethylamino group with
(1S)-2,2,2-trifluoro-1-methylethylamino group provided
the best potency. The lead compound in this series (5)
is comparable to TTI-237 in terms of cellular potency.
These compounds were evaluated in the COLO 205
cytotoxicity assay,2 and their IC50 values are shown in
Table 1. Increasing the carbon chain length of the alkyl
group on the cyanoamino nitrogen led to decrease in po-
tency (3a vs 3b and 3c). The structure–activity relation-
ship for the rest of the molecule mimics that of
[1,2,4]triazolo[1,5-a]pyrimidines.2 Replacing the 4-fluoro
group with 3-dimethylaminopropoxy group resulted in a
4-fold increase in potency (3a vs 4a). A three-methylene
tether is optimal for activity. Compound 4b, with a two-
methylene tether, is much less potent. Compound 4c,
with a four-methylene tether, is less potent than 4a,
but more potent than 4b. A possible explanation of this
result is that with a flexible four-methylene tether, the
dimethylamino group in 4c can still manage to form
Compound 5 was selected to evaluate its ability to over-
come resistance due to multidrug resistance (MDR) in
the KB lines.5 The results are shown in Table 2. Com-
pound 5 is comparable to TTI-237 in the sensitive KB
line and in the clinically more relevant KB 8.5 line. It
showed much lower levels of recognition by P-gp com-
pared with paclitaxel and vincristine.
Compound 5 shows a favorable profile as a pharmaceu-
tical agent. It has good water solubility (>100 lg/mL at
pH 7.4) and high microsomal stability (92% remaining
after incubation in nude mouse microsomes for
30 min). Mechanistic studies6 showed that compound 5
Table 1. Inhibition of COLO 205 cell proliferation by compounds 3–5
F
CF3
F
F
F
O
O
CF3
NH
(CH2)nN(CH3)2
CF3
NH
N
(CH2)nN(CH3)2
CH3
NH
N
N
N
F
R
F
N
F
N
Cl
R
R
N
Cl
N
N
Cl
CN
CN
3
CN
4
5
a
IC50 (nM)
Compound
R
n
3a
3b
CH3
CH2CH3
—
—
—
3
214
304
477
52
3c
4a
CH2CH@CH2
CH3
CH3
4b
4c
2
532
229
36
CH3
CH3
4
5
3
TTI-237b
Paclitaxel
Vincristine
31
3.3
2.6
a Determinations were made at 10 concentrations, in triplicate, and repeat values agreed, on average, within 40%.
b Tested as the HCl salt.

N. Zhang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3003–3005
3005
Table 2. Inhibition of KB, KB 8.5, and KB V1 cell proliferation by
compound 5
as TTI-237. Further modifications of the 2-cyanoamino
group are underway.
a
Compound
IC50 (nM)
KB 8.5
Ratiob
KB
KB V1
KB 8.5
KB V1
Acknowledgment
5
24
23
59
67
26
58
82
953
2.5
2.9
3.5
41.5
822
TTI-237c
Paclitaxel
Vincristine
The authors gratefully thank Dr. Joseph Ashcroft for
structural elucidations of regioisomers through exten-
sive NMR studies.
2.45
2.2
2013
2035
11
26
925
a Determinations were made at 10 concentrations, in duplicate, and
repeat values agreed, on average, within 10%.
b Ratio = IC50 on KB 8.5 or KB V1 cells/IC50 on KB cells.
c Tested as the HCl salt.
References and notes
1. Zhou, J.; Giannakakou, P. Curr. Med. Chem. Anti-Canc.
Agents 2005, 5, 65.
2. Zhang, N.; Ayral-Kaloustian, S.; Nguyen, T.; Afragola, J.;
Hernandez, R.; Lucas, J.; Gibbons, J.; Beyer, C. J. Med.
Chem. 2007, 50, 319.
behaved similarly to TTI-237. It promotes polymeriza-
tion of either MAP-rich tubulin or pure tubulin
in vitro. In tubulin binding studies,6 it competes with
[3H]vinblastine, but does not competes with [3H]colchi-
cine or [3H]paclitaxel. These findings, together with the
similarity observed in the SAR for the [1,2,4]triazolo-
[1,5-a]pyrimidine series and the 2-cyanoaminopyrimi-
dine series, strongly suggest that these two series of com-
pounds bind at the same binding site on tubulin and in
the same orientation.
3. In addition to compound 2, the other two isomers isolated
are compounds with methylation at the N-4 and the 7-
anilino nitrogen of compound 1. All these three mono-
methylated compounds showed decreased potency in the
COLO 205 assay relative to compound 1.
4. A one-step conversion of [1,2,4]triazolo[1,5-a]pyrimidines
to 2-cyanoaminopyrimidines with iodomethane and 1
equivalent of sodium hydride was reported in Pees, K.-J.;
Pfrengle, W.; Heffernan, G. WO200196314A1; Chem.
Abstr. 2001, 136, 53756. We found that our stepwise
conversion provided much higher yields in our hands.
5. Loganzo, F.; Discafani, C. M.; Annable, T.; Beyer, C.;
Musto, S.; Hari, M.; Tan, X.; Hardi, C.; Hernandez, R.;
Baxter, M.; Singanallore, T.; Khafizova, G.; Poruchynsky,
M. S.; Fojo, T.; Nieman, J. A.; Ayral-Kaloustian, S.; Zask,
A.; Andersen, R. J.; Greenberger, L. M. Cancer Res. 2003,
63, 1838.
The 2-cyanoaminopyrimidine series shows the potential
of 2-substituted pyrimidines to mimic the [1,2,4]triazolo-
[1,5-a]pyrimidine core in the TTI-237 series. We are fol-
lowing this lead by introducing various aryl and hetero-
aryl groups to the 2-position on the pyrimidine ring.
We have developed a series of 2-cyanoaminopyrimi-
dines7 as a result of our efforts to modify a series of
[1,2,4]triazolo[1,5-a]pyrimidines as anticancer agents.
The structure–activity relationship for this 2-cyanoami-
nopyrimidine series mimics that of the [1,2,4]triazolo-
[1,5-a]pyrimidine series. Compared with TTI-237, the
lead compound (5) retains in vitro activity and the capa-
bility to overcome multidrug resistance due to P-gp.
Mechanism of action studies showed that it behaved in
the same manner as TTI-237. These findings strongly
suggest that this series of 2-cyanoaminopyrimidines
binds at the same site and in the same binding mode
6. Detailed description of the mechanism study has
been provided in the Supporting Information section in
Ref. 2.
7. 1H NMR spectra of selected compounds are as follows.
Compound 2 (CDCl3): d 3.67 (s, 3H), 4.56 (q, J = 7 Hz,
2H), 6.70 (m, 2H), 7.86 (s, 1H). Compound 3a (CDCl3): d
3.47 (s, 3H), 4.19 (m, 2H), 4.83 (br s, 1H), 6.88 (m, 2H).
Compound 5 (CDCl3): d 1.33 (d, J = 7 Hz, 3H), 1.99 (m,
2H), 2.27, (s, 6H), 2.46 (t, J = 7 Hz, 2H), 3.45 (s, 3H), 4.06
(t, J = 7 Hz, 2H), 5.06 (m, 1H), 6.63 (m, 2H).