Structural studies on bioactive compounds. 39. Biological consequences of the structural modification of DHFR-inhibitory 2,4-diamino-6-(4-substituted benzylamino-3-nitrophenyl)-6-ethylpyrimidines ('benzoprims')

Article abstract of DOI:10.1021/jm040785+

Benzimidazole-N-oxide modifications of potent lipophilic dihydrofolate reductase (DHFR) inhibitors (e.g., methylbenzoprim 1 and dichlorobenzoprim 2) have been prepared by base-promoted cyclization of the nitrophenylbenzylamino groups to explore the possibility that abrogation of DHFR-inhibitory activity might reveal clues to an alternative anti-ras mechanism. Examples of the new series had only low growth inhibitory activities (GI₅₀ generally >50 μM) against colon HCT116 and lung HT29 cell lines but, unlike methylbenzoprim, this activity was unaffected by hypoxanthine/thymidine rescue.

Full text of DOI:10.1021/jm040785+

J . Med. Chem. 2004, 47, 4105-4108
4105
Brief Articles
Str u ctu r a l Stu d ies on Bioa ctive Com p ou n d s. 39.1 Biologica l Con sequ en ces of
th e Str u ctu r a l Mod ifica tion of DHF R-In h ibitor y 2,4-Dia m in o-6-(4-su bstitu ted
ben zyla m in o-3-n itr op h en yl)-6-eth ylp yr im id in es (‘ben zop r im s’)
Marianne L. Richardson, Karen A. Croughton, Charles S. Matthews, and Malcolm F. G. Stevens*
Centre for Biomolecular Sciences, School of Pharmacy, Universityof Nottingham, Nottingham, NG7 2RD, UK
Received February 10, 2004
Benzimidazole-N-oxide modifications of potent lipophilic dihydrofolate reductase (DHFR)
inhibitors (e.g., methylbenzoprim 1 and dichlorobenzoprim 2) have been prepared by base-
promoted cyclization of the nitrophenylbenzylamino groups to explore the possibility that
abrogation of DHFR-inhibitory activity might reveal clues to an alternative anti-ras mechanism.
Examples of the new series had only low growth inhibitory activities (GI50 generally >50 µM)
against colon HCT116 and lung HT29 cell lines but, unlike methylbenzoprim, this activity
was unaffected by hypoxanthine/thymidine rescue.
In tr od u ction
zyl)purine 3, which shares the 2,4-diamino-1,3-diazine
motif with dichlorobenzoprim, and is only a weak
inhibitor of rat liver DHFR (I50 252 µM), showed GI50
values of <1 µM for growth inhibition of an unspecified
“17 tumor cell lines.” Furthermore, the benzotriazolium
oxide 4, which might be considered as a cyclic variant
The nonclassical antifolate 2,4-diamino-5-(4-(N-meth-
ylbenzylamino)-3-nitrophenyl)-6-ethylpyrimidine (1,
methylbenzoprim) is representative of a family of potent
lipophilic dihydrofolate reductase (DHFR) inhibitors
(‘benzoprims’) with spectacular antitumor activity against
the methotrexate (MTX)-resistant mouse M5076 reticu-
lum cell sarcoma.2 Recently we have reported that 1 and
related structures such as the dichlorobenzylamino ana-
logue (2: dichlorobenzoprim) (I50 for rat liver DHFR )
of the benzoprim family,8 is also a relatively weak
inhibitor of rat liver DHFR (I50 ) 2.7 µM) but gives a
highly significant PCC of 0.72 relating its in vitro
activity fingerprint to the Ki-ras status of NSCL and
colon tumor cell lines.3
Because inhibition of DHFR has such a profound
effect on cellular functions in vitro and in vivo,9 design
of experiments to explore if new 2,4-diaminopyrimidines
might perturb other cancer-relevant molecular targets
relies on reversal experiments applying exogenously the
products of the inhibited folate mechanism. In a me-
dicinal chemistry approach to address this question we
have redesigned both substructures of the benzoprim
pharmacophore to ablate DHFR-inhibitory potency: (a)
by effecting a base-promoted intramolecular cyclization
between the o-nitrophenyl substituent and the benzyl-
amino residues to generate benzimidazole-N-oxides,10
analogous in structure to the lead benzotriazole-N-oxide
4; and (b) by removing the 4-amino and 6-ethyl groups
to furnish 4-desaminobenzoprims of reduced basicity
and lipophilicity compared to the lead benzoprims. Both
these features are required for tight binding inhibition
of DHFR.11
0.008 µM) demonstrate selective in vitro growth-inhibi-
tory activities in cell lines of the non small cell lung
(NSCL) and colon cancer subpanels of the National
Cancer Institute (NCI) 60-cell panel which harbor
mutations in codon 12 of the Ki-ras gene.3 Significant
Pearson correlation coefficients (PCCs) for methylben-
zoprim (0.77) and dichlorobenzoprim (0.79) in the NCI
COMPARE algorithm4,5 link the GI50 values of these
compounds with the ras status (wild or mutated) of
NSCL and colon cancer cell lines. Moreover, compound
2 has potent inhibitory activity against the CCRF-CEM
human T-cell leukemia growing in athymic mice: this
tumor bears a mutation in codon 12 of the Ki-ras 2 gene
(Gly to Asp) and is intrinsically resistant to MTX.3
6
Other observers have concluded that lipophilic 2,4-
diaminopyrimidine DHFR inhibitors might have an
alternative nonfolate mechanism. Gangjee and col-
leagues7 reported that 2,6-diamino-8-(3,4-dichloroben-
Ch em istr y
Several of the benzoprims required for this work have
been reported in our earlier work from the interaction
* Corresponding author. E-mail: malcolm.stevens@nottingham.ac.uk.
Phone: +44 (0)1159513414; Fax: +44 (0)1159513412.
10.1021/jm040785+ CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/29/2004

4106 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Brief Articles
Ta ble 1. Yieldsa and Melting Pointsb of 2,4-Diamino-5-
(4-substituted benzylamino-3-nitrophenyl)-6-ethylpyrimidines,
2-Amino-5-(4-substituted benzylamino-3-nitrophenyl)pyrimi-
dines, 5-(2,4-Diamino-6-ethylpyrimidin-5-yl)-2-(substituted
phenyl)-1H-benzimidazole-3-oxides, and 5-(2-Aminopyrimidin-
5-yl)-2-(substituted phenyl)-1H-benzimidazole-3-oxides
Sch em e 2a
compound yield (%) mp (°C) compound yield (%) mp (°C)
7
9
45
44
38
40
69
75
92
75
95
60
98
61
65
54
99c
72
61c
61
84
260
226
256
234
36
37
38
39
40
41
42
43
44
45
46
47
48
50
51
52
53
54
55
77
76
91
63
82
60
84
67
83
71
55
66
77
81
96
89
95
89
77
233
247
254
192
229
229
285
211
241
225
221
249
240
290d
302d
271d
302d
301d
220d
13
15
17
19
20
21
22
23
24
25
26
27
28
29
30
31
35
219
191d
273d
195d
212d
174d
174d
246d
262d
256d
232d
184d
190d
187
216
a
For details of synthetic method, see Experimental Section.
For details of other physical data (IR, 1H NMR, 13C NMR, and
b
mass spectra), see Supporting Information. c DMSO solvent used
d
a
in synthesis. Melts with decomposition.
Reagents and conditions: (a) HNO3-H2SO4; (b) 50 °C, 2 h;
34, substituted benzylamine, reflux, 5 h; (c) NaOH in 2-ethoxy-
ethanol, reflux 5 h, then neutralize with HCl.
of 2,4-diamino-5-(4-chloro-3-nitrophenyl)-6-ethylpyrimi-
dine (5; ‘nitropyrimethamine’) and substituted benzyl-
amines.2 Yields and melting points of new benzoprims,
4-desaminobenzoprims, and their benzimidazole-N-
oxide cyclization products are listed in Table 1. The
model compound N-benzyl-N-methyl-2-o-nitroaniline
does not cyclize in base12 and attempts to cyclize
methylbenzoprim 1 to the corresponding benzimidazole
oxide 18 using inorganic bases NaOH or K2CO3 in hot
2-ethoxyethanol or DMSO were similarly unsuccessful.
In contrast the benzylamines 2, 6-17 all cyclized to
benzimidazole oxides 19-31 (Scheme 1) in yields > 60%
(Table 1). Generally, cyclization was complete within 2
h with NaOH but required 5 h using K2CO3. Attempts
to prepare the benzimidazole oxides 28 and 30 in
2-ethoxyethanol containing K2CO3 directly from 5, and
the more weakly basic benzylamines bearing powerful
electron-withdrawing F or CF3 groups led to the isola-
tion of the ether 32 (∼50%), also obtained (65%) when
nitropyrimethamine was boiled in 2-ethoxyethanol con-
taining base. To avoid the possibility that the substi-
tuted benzylamino groups of 14 and 16 might suffer
nucleophilic displacement under cyclization conditions
in 2-ethoxyethanol, compounds 28 and 30 were most
efficiently synthesized using NaOH in DMSO as solvent.
The starting point for the synthesis of 4-desami-
nobenzoprims was 2-amino-5-(4-chlorophenyl)pyrimi-
dine 33 which we have prepared recently by a Suzuki
reaction between 2-amino-5-bromopyrimidine and 4-chlo-
robenzeneboronic acid.13 Nitration of 33 in a HNO3-
H2SO4 mixture at 45-50 °C gave 2-amino-5-(4-chloro-
3-nitrophenyl)pyrimidine 34 which was reacted with
neat benzylamines to furnish the new series 35-48.
Representative samples were then cyclized by base to
afford the oxides 50-55 (Scheme 2 and Table 1). Again,
the N-methylbenzylamine 35 could not be cyclized to a
benzimidazole-N-oxide, 49.
Unlike the highly crystalline orange-red benzoprims,
Sch em e 1a
a
Reagents and conditions: (a) substituted benzylamine, reflux, 5 h; (b) K2CO3 in 2-ethoxyethanol, reflux, 42 h; (c) NaOH in
2-ethoxyethanol, reflux 5 h, then neutralize with HCl; (d) NaOH in DMSO, 80 °C, 2 h, then neutralize with HCl.

Brief Articles
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16 4107
Ta ble 2. GI50 Values (µM)a of Methotrexate (Mtx),
Trimetrexate (TMX), Methylbenzoprim (1), Nitropyrimethamine
(5), and Novel Compounds against Colon HCT-116 and HT29
Cells ( Hypoxanthine-Thymidine (HT) Rescueb
completely by HT rescue has suggested to us that
methylbenzoprim may have an alternative non-folate
mechanism.3 Data for the 2-methoxy-derivative 9 showed
a similar partial rescue by HT (Table 2). In contrast the
GI50 values against HCT-116 and HT29 for all the new
compounds, in their acyclic (34, 35, 42, and 44) or cyclic
modifications (26, 28, 50, 51, and 53), were uninfluenced
by HT. Specimen dose-response curves for methylben-
zoprim 1 and three compounds (26, 42, and 53) repre-
sentative of the new structural types against HCT-116
cells ( HT rescue are included in Supporting Informa-
tion. These data confirm that the chosen structural
modifications abrogate DHFR-inhibitory properties lead-
ing to loss of growth-inhibitory activity but do not reveal
clues to a novel cancer-relevant mechanism. In cor-
roboration, flow cytometric analysis of HCT-116 and
HT29 cells treated with compounds 26, 42, and 53 at
their respective GI50 values (from Table 2) revealed no
significant cell cycle perturbations (data not shown).
compound HCT116 HCT116 + HT
HT29
HT29 + HT
MTX
TMX
1
5
9
26
28
34
35
42
44
50
51
53
0.01
0.01
0.08
0.25
0.08
9.0
>50
0.07
0.01
0.48
3.8
>50
21
9.2
40
11.6
>50
39
>50
17
25
38
19
7.4
38
7.5
9.0
22
>50
>50
5
1.5
>50
46
>50
14
17
17
>50
>50
3
48
47
41
>50
>50
7
>50
>50
9
>50
>50
17
>50
>50
15
a
MTT assays were carried out following 4 days exposure to
increasing concentrations of drug. Results are the mean of three
b
experiments.
(20 µM).
H ) hypoxanthine (100 µM); T ) thymidine
Exp er im en ta l Section
the benzimidazole-N-oxides were off-white solids with
a chalky consistency which decomposed on attempted
crystallization. The compounds could be purified by
precipitation from DMSO with water and, like many
2,4-diaminopyrimidines, did not give reliable elemental
analyses because of partial hydration. Compounds were
characterized by NMR and HRMS analysis. For ex-
Ch em ica l Syn th esis. Melting points (uncorrected) were
measured using a Gallenkamp melting point apparatus.
2,4-Dia m in o-5-(4-ch lor o-3-n itr op h en yl)-6-eth ylp yr im i-
d in e (5; n itr op yr im eth a m in e). This compound was prepared
by a published method.14
2-Am in o-5-(4-ch lor o-3-n it r op h en yl)p yr im id in e (34).
2-Amino-5-(4-chlorophenyl)pyrimidine (2.402 g, 11.7 mmol)13
was added to a stirred mixture of nitric (d, 1.42, 10 mL) and
sulfuric acid (10 mL) over 20 min with the temperature
maintained below 50 °C. The yellow syruplike solution was
heated at 45-50 °C for 2 h and then stirred at room temper-
ature for 1.5 h. The mixture was poured slowly into an ice-
concentrated ammonia mixture (to >pH 10). The pale yellow
product (84%) was collected, washed with water, and dried at
40 °C. The pyrimidine recrystallized from 50% ethanol as
yellow crystals, mp 260 °C. Anal. Calcd for C10H7ClN4O2: C,
H, N.
1
ample, in the H NMR spectra the benzylic protons of
the benzoprims (at ∼δ 4.5-4.6) are generally coupled
to the benzylamine NH protons: these features are
absent in the spectra of the oxides (see Supporting
Information).
Biologica l Resu lts a n d Discu ssion
Gr ow t h -In h ib it or y Act ivit y of Com p ou n d s
a ga in st Hu m a n Colon Cell Lin es in Vitr o. The
inhibitory effects (GI50 values) of several new com-
pounds were measured against the colon cell lines HCT-
116, which bears a GGC (gly) to GAC (asp) mutation in
Ki-ras codon 13, and HT29 with wild type ras, in a 4-day
MTT assay. To ascertain if inhibitory mechanisms
differed from those of the classical DHFR inhibitor
methotrexate (MTX) and the lipophilic antifolate tri-
metrexate (TMX), methylbenzoprim 1, nitropyrimeth-
amine 5, and several new compounds were also evalu-
ated in the presence/absence of hypoxanthine-thymidine
(HT) rescue (Table 2). Overall there was a 5000-fold
range in potencies, and compounds were more growth-
inhibitory toward the mutated HCT-116 line. The most
active agents were MTX and TMX. Of the compounds
with an o-nitro substituent, the most growth-inhibitory
against HCT-116 were the lipophilic antifolates meth-
ylbenzoprim 1, nitropyrimethamine 5, and the novel
2-methoxy-substituted benzoprim 9, a close analogue of
1. Removal of the 4-amino and 6-ethyl groups in
compounds 34, 35, 44 had a significant dyschemothera-
peutic effect in comparison to the foregoing agents with
only 42 showing activity in the low micromolar range.
Similarly, compounds bearing the benzimidazole-N-
oxide variation 26, 28, 50-51 displayed only weak
growth-inhibitory properties against both cell lines with
53 being the most active but still approximately 100-
fold less potent than methylbenzoprim 1.
Gen er a l Meth od for th e P r ep a r a tion of 2,4-Dia m in o-
5-(4-su bstitu ted ben zyla m in o-3-n itr op h en yl)-6-eth ylp y-
r im id in es a n d 2-Am in o-5-(4-su bstitu ted ben zyla m in o-3-
n itr op h en yl)p yr im id in es. A suspension of 2,4-diamino-5-
(4-chloro-3-nitrophenyl)-6-ethylpyrimidine (10.0 g, 34 mmol)14
or 2-amino-5-(4-chloro-3-nitro-phenyl)pyrimidine (8.52 g, 34
mmol) in the appropriate pure benzylamine (30 mL) was
heated under reflux for 5 h. The red colored solutions were
cooled and diluted with water (for water-miscible amines) or
ether (for water immiscible amines) to liberate the required
compounds. All products were recrystallized from aqueous
2-ethoxyethanol. Compounds 1, 2, 6, 8, 10-12, 14, and 16 have
been prepared previously.2 Yields and melting-points of new
1
compounds are assembled in Table 1. IR, H NMR, 13C NMR,
and mass spectroscopic data are included in Supporting
Information.
Gen er a l Meth od for th e Syn th esis of 5-(2,4-Dia m in o-
6-eth ylp yr im id in -5-yl)-2-(su bstitu ted p h en yl)-1H-ben z-
im id a zole-3-oxid es a n d 5-(2-Am in op yr im id in -5-yl)-2-
(su b st it u t ed p h en yl)-1H -b en zim id a zole-3-oxid es. 2,4-
Diamino-5-(4-substituted benzylamino-3-nitrophenyl)-6-ethyl-
pyrimidine (0.8 mmol) or 2-amino-5-(4-substituted benzyl-
amino-3-nitrophenyl)pyrimidine (0.8 mmol) and sodium hy-
droxide (8 mmol) were heated under reflux in 2-ethoxyethanol
(5 mL) for 5 h. On cooling, the reaction mixture was diluted
with water and neutralized with 0.5 M HCl. The precipitated
product was collected by filtration and dried. Yields and
melting-points of new compounds are assembled in Table 1.
IR, 1H NMR, 13C NMR, and mass spectroscopic data are
included in Supporting Information.
2,4-Dia m in o-5-(4-eth oxyeth yloxy-3-n itr op h en yl)-6-eth -
ylp yr im id in e (32). (i) 2,4-Diamino-5-(4-chloro-3-nitrophenyl)-
6-ethylpyrimidine (1.005 g, 3.42 mmol), potassium carbonate
The fact that the antitumor activity of methylben-
zoprim 1, unlike that of MTX, cannot be reversed

4108 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 16
Brief Articles
(0.95 g, 6.84 mmol), and 2-ethoxyethanol (10 mL) were heated
under reflux for 42 h. The reaction mixture was cooled to room
temperature and neutralized with 0.5 M HCl. The yellow solid
was collected by filtration and dried (65.2%), mp 206-207 °C.
(ii) 2,4-Diamino-5-(4-chloro-3-nitrophenyl)-6-ethylpyrimidine
(1.00 g; 3.42 mmol), 4-fluorobenzylamine [or 4-(trifluorometh-
yl)benzylamine] (3.42 mmol), and potassium carbonate (0.95
g; 6.84 mmol) were heated under reflux in 2-ethoxyethanol (10
mL) for 60 h. The reaction mixture was cooled to room tem-
perature and then neutralized with 0.5 M HCl. The precipitate
which formed was collected by filtration and dried (50%) and
was identical (IR, 1H NMR) to the sample of 2,4-diamino-5-
(4-ethoxyethyloxy-3-nitrophenyl)-6-ethylpyrimidine 32, pre-
pared above.
Biologica l In vestiga tion s. Dr u g Solu tion s. Drug stock
solutions were all made up to10 mM with compounds dissolved
in the following vehicles: MTX, TMX, methylbenzoprim
(MBP), and all new compounds in DMSO; hypoxanthine (H)
in 10 mM NaOH; thymidine (T) in distilled water. All solutions
were sterile filtered and stored at -20 °C.
Cell Cu ltu r e. Colon HCT-116 (ATCC No. CCL-247) and
HT29 (ATCC No. HTB-38) cell lines were cultured in RPMI
1640 medium with L-glutamine (Gibco BRL Life Technologies)
supplemented with 10% heat-inactivated (55 °C for 1 h) fetal
bovine serum (FBS). All cells were maintained at 37 °C in an
atmosphere of 5% CO2 and 95% air. Cells were mycoplasma
free and passaged 2-3 times weekly; the medium aspirated
off, 1-2 mL of trypsin/EDTA added and left for 2-5 min until
the cells had become detached before addition of 5 mL medium
to inactivate the trypsin. Cells were split into T25 flasks
(Costar) containing 7-10 mL medium at the desired concen-
tration. Cells were maintained in a Leec incubator and all cell
culture processes were carried out in a MDH class II micro-
biological safety cabinet with a laminar flow system swabbed
before each use with 70% ethanol in distilled water.
Gr ow th In h ibition Assa ys. Following trypsinisation cells
were placed in 5 mL of nutrient medium and syringed to
remove cell clumps. Cells were counted using a Neubauer
haemocytometer and plated in 96-well plates (Nunclon) at
appropriate densities for the rate of cell division and the
duration of the experiment. Cells were allowed to attach (4 to
24 h) prior to addition of drug to the appropriate concentration
bringing the final volume in each well up to 200 µL. Where
present H was added to a final concentration of 100 µM and T
to 20 µM. A time zero plate was made up to 200 µL in each
well by the addition of medium to provide the initial optical
density reading. Following incubation at 37 °C in an atmo-
sphere of 5% CO2 and 95% air for the duration of the
experiment (4 days), 50 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT; 2 mg/mL in PBS) was
added to each well containing 200 µL of medium. Following a
4 h incubation period, the MTT/medium was aspirated from
each well and 125 µL of DMSO:glycine buffer (4:1) was added
to each well. The plates were then shaken to dissolve all the
formazan crystals formed and read at 550 nm on an Anthos
plate reader (Anthos Labtech Instruments). Results, expressed
as GI50 values, were analyzed using the Deltasoft 3 computer
program (BioMetallics Inc., PO Box 2251, Princetown, NJ
08543).
Su p p or tin g In for m a tion Ava ila ble: C, H, N analyses
for compound 34; IR, 1H NMR, 13C NMR, and mass spectra
for compounds 7, 9, 13, 15, 17, 19-32, 34-48, 50-55; dose-
response curves for compounds 1, 26, 42, 53 against human
HCT-116 cells in the absence or presence of hypoxanthine (H)
and thymidine (T) rescue. This material is available free of
charge via the Internet at http://pubs.acs.org.
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J M040785+