Synthesis and biological evaluation of 5-substituted O4- alkylpyrimidines as CDK2 inhibitors

Article abstract of DOI:10.1039/b925481a

CDK2 inhibitory structure-activity relationships have been explored for a range of 5-substituted O⁴-alkylpyrimidines. Variation of the 5-substituent in the 2,6-diaminopyrimidine series confirmed the 5-nitroso substituent as optimal, and showed that 5-formyl and 5-acetyl substituents were also tolerated at this position. A series of O⁴-alkyl-N ²-aryl-5-substituted-6-aminopyrimidines revealed interesting structure-activity relationships. In the 5-nitroso series, the optimum O ⁴-alkyl substituents were cyclohexylmethyl or sec-butyl, combined with a 2-sulfanilyl group. By contrast, in the N²-arylsulfonamido-5- formyl series, the cyclohexylmethyl compound showed relatively poor activity compared with the sec-butyl derivative (22j, (R)-4-(4-amino-6-sec-butoxy-5- formylpyrimidin-2-ylamino)benzenesulfonamide; CDK2 IC₅₀ = 0.8 nM). Similarly, in the N²-arylsulfonamido-5-(hydroxyiminomethyl) series the O⁴-sec-butyl substituent conferred greater potency than the cyclohexylmethyl (23c, (rac)-4-(4-amino-6-sec-butoxy-5-(hydroxyiminomethyl) pyrimidin-2-ylamino)benzenesulfonamide; CDK2 IC₅₀ = 7.4 nM). The 5-formyl derivatives show selectivity for CDK2 over other CDK family members, and are growth inhibitory in tumour cells (e.g.22j, GI₅₀ = 0.57 μM).

Full text of DOI:10.1039/b925481a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological evaluation of 5-substituted O4-alkylpyrimidines as
CDK2 inhibitors†
Francesco Marchetti,a Ce´line Cano,a Nicola J. Curtin,b Bernard T. Golding,c Roger J. Griffin,a
Karen Haggerty,a David R. Newell,b Rachel J. Parsons,a Sara L. Payne,a Lan Z. Wangb and Ian R. Hardcastle*a
Received 3rd December 2009, Accepted 19th February 2010
First published as an Advance Article on the web 18th March 2010
DOI: 10.1039/b925481a
CDK2 inhibitory structure–activity relationships have been explored for a range of 5-substituted
O4-alkylpyrimidines. Variation of the 5-substituent in the 2,6-diaminopyrimidine series confirmed the
5-nitroso substituent as optimal, and showed that 5-formyl and 5-acetyl substituents were also tolerated
at this position. A series of O4-alkyl-N2-aryl-5-substituted-6-aminopyrimidines revealed interesting
structure–activity relationships. In the 5-nitroso series, the optimum O4-alkyl substituents were
cyclohexylmethyl or sec-butyl, combined with a 2-sulfanilyl group. By contrast, in the
N2-arylsulfonamido-5-formyl series, the cyclohexylmethyl compound showed relatively poor activity
compared with the sec-butyl derivative (22j, (R)-4-(4-amino-6-sec-butoxy-5-formylpyrimidin-
2-ylamino)benzenesulfonamide; CDK2 IC50 = 0.8 nM). Similarly, in the N2-arylsulfonamido-5-
(hydroxyiminomethyl) series the O4-sec-butyl substituent conferred greater potency than the
cyclohexylmethyl (23c, (rac)-4-(4-amino-6-sec-butoxy-5-(hydroxyiminomethyl)pyrimidin-2-
ylamino)benzenesulfonamide; CDK2 IC50 = 7.4 nM). The 5-formyl derivatives show selectivity for
CDK2 over other CDK family members, and are growth inhibitory in tumour cells (e.g. 22j, GI50
0.57 mM).
=
of mitosis. Finally, CDK1 activation by the B-cyclins drives the
cells through mitosis. On this basis, we and many other groups
have sought to develop potent and specific inhibitors of CDKs, in
particular CDKs 1, 2 and 4, as cancer therapeutic agents.
More recently, however, a series of genetic studies in mice,
in which specific CDKs have been disrupted, has demonstrated
that CDK2, CDK4 and CDK6 are not essential for the cell
cycle in most cells, and that the knockout mice grow with few
abnormalities.2,5 By contrast, the CDK1 knockout is lethal at
the two-cell embryo stage. Further experiments with multiple
CDK knockouts, showed that specific combinations of CDKs are
required to allow differentiation of specialised cell types within the
developing embryo, suggesting an additional role for the cyclin-
dependent kinases.
The classical model of CDK function in the cell cycle suggested
that selective small-molecule CDK inhibitors could be used to
restore checkpoint control in many cancers. This suggestion
resulted in a large effort from a number of groups worldwide to
discover novel therapeutics based on CDK inhibition.6 Despite
unpromising results in clinical trials from the first generation of
CDK inhibitors, which lacked potency and selectivity, a number
of second generation, ATP-competitive, small-molecule inhibitors
are currently in clinical evaluation (Chart 1). Of these, only
P1446A-05 (structure not disclosed) is described as selective for a
single CDK (CDK4); the others inhibit multiple CDKs, including
the essential CDK1, plus CDK7 which regulates the cell cycle
CDKs, and CDK9 which is involved in transcription.
Introduction
Disregulation of the cell-cycle resulting in unscheduled cell
division, coupled with genomic and chromosomal instability, is a
common feature of cancer.1 The cell cycle is regulated by the cyclin-
dependent kinase (CDK) family of enzymes and their partner
cyclins, and mutations resulting in abnormal CDK activity have
been found in many tumours.2
The ‘classical’ model of the cell-cycle, developed over the past
20 years, proposed that specific CDK/cyclin combinations were
required to be activated to allow progression through checkpoints
leading to entry into the critical phases of DNA-synthesis and
mitosis. Activation of CDK4 and CDK6 by cyclins D1, D2, and
D3 allows progression from G1 into S-phase. The synthesis of
cyclin E1 and E2 is also initiated by activation of CDK4 and
CDK6, which in turn activates CDK2. Phosphorylation of the
Rb protein requires CDK2, thus allowing progression through
the early S-phase. Early experiments showed that blocking CDK2
by mutation or antibodies resulted in the inhibition of the cell
cycle in tumour cells, suggesting that CDK2 activity was essential
for progression through G1 to S-phase.3,4 Activation of CDK2
by cyclin A2 occurs during the G2-phase and permits the start
aNorthern Institute for Cancer Research, Bedson Building, Newcastle
University, Newcastle Upon Tyne, UK NE1 7RU. E-mail: I.R.Hardcastle@
ncl.ac.uk; Tel: +44 191 222 6645
bNorthern Institute for Cancer Research, Paul O’Gorman Building, Medical
School, Framlington Place, Newcastle University, Newcastle Upon Tyne,
UK NE2 4HH
We have described previously two series of potent and selective
CDK2 inhibitors based on either a purine scaffold e.g. (1)7,8 or the
purine-mimetic 5-nitrosopyrimidine scaffold (2).9 In this study,
we sought to widen the structure activity relationships (SAR) for
cSchool of Chemistry, Bedson Building, Newcastle University, Newcastle
Upon Tyne, UK NE1 7RU
† Electronic supplementary information (ESI) available: Additional exper-
imental details. See DOI: 10.1039/b925481a
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Scheme 1 Reagents and conditions: a) Na, ROH, D; or NaH, ROH,
DMSO, D; b) AcOH, NaNO2, H2O, D.
diaminopyrimidine was heated with a sodium alkoxide in DMSO
to give the required 4-alkoxypyrimidine (3a–f). 5-Nitrosation
was achieved by heating 3a–f with sodium nitrite in acetic acid
giving the corresponding 5-nitrosopyrimidines (4a–f) in moderate
yields. Reasoning that the low yields may be due to hydrolysis of
the 4-alkoxy group under the acidic conditions, a non-aqueous,
less acidic medium was sought. The use of sodium nitrite with
1.5 equiv. trifluoroacetic acid in trifluoroethanol gave much
improved yields.
A
range of 5-substituents was introduced into 4-
cyclohexylmethoxy-2,6-diaminopyrimidine (3f) using the methods
shown in Scheme 2. The 5-bromo derivative (5) was prepared in
good yield by bromination with N-bromosuccinimide.10 Similarly,
the 5-iodo compound (6) was prepared using N-iodosuccinimide.
Bromopyrimidine 5 was used as the substrate for Suzuki-Miyura
cross-coupling reactions with the appropriate aryl- or heteroaryl-
boronic acid. Elevated temperatures of 170 ◦C, achieved with
microwave heating, were essential for the synthesis of compounds
(7a–d), albeit in poor yields. Direct cyanation of bromopyrimidine
5 was achievable, but the purification was complicated by the
presence of copper complexes. As an alternative, the N,N-diacetyl
compound (8) was prepared by heating 5 in acetic anhydride.11
Treatment of 8 with copper(I)cyanide and ethane-1,2-diamine
in DMF gave the 5-cyano derivative (9) with concomitant loss
of the acetyl groups. The cyano group was converted into the
imine (10) by treatment of 9 with methylmagnesium bromide at
100 ◦C in THF. Hydrolysis of 10 to the ketone (11) occurred
readily on treatment with dilute HCl.12 For the preparation of
the 5-formyl derivative (12), the 5-cyano compound (9) was
Chart 1
the pyrimidine series. In particular, we intended to improve the
drug-like characteristics of this series by replacing the lipophilic
cyclohexylmethoxy group with smaller O4-substituents and by
seeking a replacement for the 5-nitroso group, with which there
may be toxicity problems. In this paper, we report the discovery of
highly potent CDK2 inhibitors, including one compound with
sub-nanomolar potency, which retain selectivity against other
CDKs.
Results and discussion
Chemical synthesis
A series of O4-alkoxy-5-nitrosopyrimidines was prepared using
the method described previously (Scheme 1).9 4-Chloro-2,6-
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Scheme 2 Reagents and conditions: a) NBS, AcOH, 60 ◦C; b) ArB(OH)2, Pd(PPh3)4, K2CO3, H2O, DME, MW, 170 ◦C; c) NIS, AcOH, 60 ◦C; d) Ac2O,
D; e) CuCN, DMF, ethane-1,2-diamine; f) MeMgBr, THF, 0 ◦C then 100 ◦C; g) HCl, H2O, THF; h) H2SO4, H2O, Pd/C, H2 (1 atmos.).
reduced with hydrogen/palladium on charcoal with trapping
of the intermediate imine with aqueous sulfuric acid.13 The
4-cyclohexylmethoxy-5-nitropyrimidine (14) was prepared from
4-chloro-2,6-diaminopyrimidine (Scheme 3) by nitration with
fuming nitric acid in sulfuric acid to give (13), followed by reaction
with sodium cyclohexylmethoxide in t-butanol.14
Oxidation of 16a–c to the corresponding butylsulfones (17a–
c) with m-chloroperbenzoic acid (mCPBA) was followed by
displacement of the butanesulfonyl group with the appropriate
aniline to give 18a–c.15 Subsequent nitrosation at the 5-position
gave the desired compounds (19a–e).
An alternative strategy was required for the synthesis of
the 2-arylamino-5-formylpyrimidines (Scheme 5). 4-Amino-2,6-
dihydroxypyrimidine was reacted with phosphorous oxychloride
and DMF to give the 2,6-dichloro-5-formyl pyrimidine (20). The
N2-aryl substituent was introduced using the previously optimised
conditions for SNAr displacements at the 2-position of pyrimidines
using TFE as solvent with TFA to give the corresponding 4-
chloro derivatives (21a–c).15 Displacement of the 4-chloro group
with the required sodium alkoxide gave the O4-alkoxy substituted
compounds (22a–l) in moderate yields. In the case of the benzoate
derivative (22e), the carboxylic acid was protected as its neopentyl
ester for the alkoxide displacement and removed during the
basic work up. Selected 5-formyl compounds were converted into
the respective oximes (23a–c) on treatment with hydroxylamine
hydrochloride in pyridine.
Scheme 3 Reagents and conditions: a) HNO3, H2SO4, 35 ◦C; b) NaOtBu,
tBuOH, cyclohexylmethanol, 30 ◦C.
The corresponding O4-ethoxy- and O4-sec-butoxy- derivatives
were prepared for comparison with the O4-cyclohexylmethoxy
compound (19a) using the method described previously
(Scheme 4).9 Alkylation of pyrimidinone (15) under Mitsunobu
conditions gave the O4-alkoxy derivatives (16a–c) good yields.
Scheme 4 Reagents and conditions: a) ROH, PPh3, DEAD, THF; b) mCPBA, DCM; c) ArNH2, TFA, TFE, D; d) AcOH, NaNO2, H2O, D.
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Scheme 5 Reagents and conditions: a) DMF, POCl3, 105 ◦C; b) ArNH2, TFA, TFE, D; c) ROH, NaH, THF, D; d) NH2OH.HCl, EtOH, pyridine.
Table 1 Inhibition of CDK2 by O4-substituted-2,6-diamino-5-
nitrosopyrimidines
Compound
R
IC50/mM
4a
4b
4c
CH3
CH2CH3
79
50
16 0.3
6
1
4d
22a
Fig. 1 Binding mode of NU6027 with CDK2.
4e
4f
-(CH2)6CH3
22%b
2.2 0.5c
is considered to be unacceptable for pharmaceuticals as it may
form toxic and genotoxic metabolites in vivo. For this reason, a
group of alternative 5-substituents was explored (Table 2). The
5-cyano substituent (9), which has similar electronic properties
to the nitroso group but is unable to form an intramolecular
hydrogen bond due to its linearity, was 30-fold less potent than
the parent 5-nitrosopyrimidine 4f. The 5-nitro substituent (14)
showed only a 4-fold loss of potency, where in this case an
intramolecular hydrogen bond can be formed. The 5-(2-furyl) and
5-(2-thiophenyl) derivatives (7b and 7c) were both 25-fold less
active than 4f, indicating that any hydrogen bonding from a furan
oxygen lone pair was insignificant and that hydrophobic bulk at
this position is unfavourable. The 5-bromo, 5-iodo and 5-phenyl
derivatives (5, 6, and 7a) lacked both solubility and potency. The
5-acetyl and 5-formyl derivatives 11 and 12, respectively, were
both 10-fold less active than 4f, whereas the imine derivative 10
suffered a 50-fold drop in potency. Crystal structure analysis of
the 5-formyl derivative 12 bound to CDK2 failed to show an
intramolecular hydrogen bond, although splitting of the 6-NH2
proton resonances in 12 in the proton NMR spectrum indicates
that hydrogen bonding occurs in DMSO solution.18
a n = 1; b % inhibition at 10 mM; c ref.18
SAR discussion
All compounds were evaluated for CDK inhibitory activity using
published procedures.8,16 Firstly, variation of the O4-alkoxy sub-
stituent was investigated with the simple 2,6-diaminopyrimidine
scaffold. The results show that small O4-alkoxy substituents confer
lower potency compared with the cyclohexylmethoxy substituent
(Table 1). These results are consistent with previous studies in the
O6-alkylguanine series17 and the O4-alkoxypyrimidine series.18 The
efficacy of the cyclohexylmethoxy substituent has been explained
by examining the X-ray structure of NU6027 bound to CDK2,
which shows the cyclohexyl group occupying the hydrophobic
entrance to the ribose binding pocket. The 2-amino group extends
close to the hydrophobic selectivity pocket of the enzyme (Fig. 1).
We have previously shown that the 5-nitroso substituent is
important for CDK inhibitory activity by forming an intramolec-
ular H-bond with the 6-amino group, which orientates the
nitrogen in a pseudo-purine geometry.19 The nitroso substituent
A series of N2-aryl compounds was prepared to investigate
structural variations within the O4-cyclohexylmethoxy-N2-aryl-5-
nitrosopyrimidines (Table 3).9 The aryl substituents were limited
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Table 2 Inhibition of CDK2 by 5-substituted-O4-cyclohexylmethoxy-
2,6-diaminopyrimidines
Table 3 CDK2 inhibition for O4-substituted-N2-aryl-5-substituted-6-
aminopyrimidines
Compound
X
IC50/mM
Compound
R
Ar
X
IC50/nM
1.0 0.3b
4f
5
6
7a
7b
NO
Br
I
2.2 0.5d
2%b
19a
NO
33%c
48%c
56.3
Ph
19b
19c
19d
NO
NO
NO
215 50b
110
7c
55
9
10
CN
61 10
92a
CH2CH3
93a
11
23a
19e
NO
1.6
12
14
24a,d
22a
22b
22c
22d
22e
22f
2900
1900
1800
225
NO2
8.3 5.6
CH2CH3
a n = 1; b percent inhibition at 10 mM; c percent inhibition at 100 mM;
d ref.18
to the 4-sulfonamide, 4-methoxy, and the previously unreported
4-carboxylic acid, as representative examples. The 5-substituents
explored were the nitroso, formyl, and the methyliminyl groups.
The 4-cyclohexylmethoxy-2-(4-methoxybenzyl) derivatives, 19b
and 22a, were 200-fold and 60-fold less active than the correspond-
ing 5-nitroso- and 5-formyl-2-sulfanilylpyrimidine derivatives, 19a
and 22k, respectively. The dramatic fall in potency is similar to the
>100 fold reduction in activity seen in the corresponding purine
series, and is consistent with the key hydrogen bond interactions
made by the sulfonamide within the ATP-binding domain of
CDK2.8 Variation was also made at the O4-position with the
introduction of smaller alkyl substituents. In the 2-sulfanilyl-5-
nitroso series, the 4-ethoxy derivative 19d was approximately 100-
fold less active than the parent 19a. By contrast, the racemic sec-
butoxy derivative 19e was equipotent with 19a. A comparison of
these results with those observed for the 2-aminopyrimidine series
(4b, 4d, and 4f), again suggests that the 2-sulfanilyl group is a
major determinant of CDK2 inhibitory activity for this series.
In the 2-(4-methoxyanilino)-5-formyl series (22a–d), the sec-
butoxy derivative 22d was >100-fold less active than the 5-nitroso
derivative 19e, consistent with results seen in the 2,6-diamino series
(4f and 12). The 4-ethoxy and 4-iso-propoxy derivatives, 22b and
22c, showed a modest improvement in activity compared with 22a,
and surprisingly, the racemic sec-butoxy compound 22d showed
comparable activity to the 4-cyclohexylmethoxy-5-nitroso deriva-
tive 19b. In the 2-sulfanilyl-5-formyl series (22f–l), the 4-ethoxy-
610
CH2CH3
49
22g
22h
22i
22j
26
3.8
13.1
0.77
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Table 3 (Contd.)
butoxy-5-formylsulfonamide 22j retained excellent selectivity for
CDK2 over CDK4/cyclinD and CDK7/cyclinH, but displayed
only 10-fold selectivity against CDK1/cyclinB and 50-fold selec-
tivity vs. CDK9/cyclinT. The same compounds were evaluated
for growth inhibitory activity using an Rb-positive SKUT1B
cancer cell line.5 As has been demonstrated previously, the CDK2
inhibitors showed only modest growth inhibitory activity (GI50
~1 mM) in comparison to their potency as inhibitors of CDK2
(IC50 ~1 nM).
Compound
R
Ar
X
IC50/nM
49
22k
Conclusions
22l
92b
In summary, we have synthesised a series of 5-substituted O4-
alkylpyrimidine CDK2 inhibitors, with a view to investigating
structure activity relationships for this chemotype. The results
are consistent with those determined previously for the analo-
gous purine series, and support the key role played by the 2-
sulfanilyl substituent. In addition, we have demonstrated that
a 4-sec-butoxy substituent can replace the cyclohexylmethoxy
group at this position without loss of potency, and evidence of
stereospecificity of binding was observed. At the pyrimidine 5-
position, a small electron withdrawing group capable of making an
intramolecular hydrogen bond with the 6-amino group is favoured.
Of the substituents explored the 5-formyl was preferred. Thus, the
combination of 4-(R)-sec-butoxy-5-formyl-2-sulfanilylpyrimidine
conferred excellent CDK2 inhibitory activity (22j; IC50 = 0.8 nM)
and is one of the most potent CDK2 inhibitors reported to date.
23a
23b
8300
117
23c
7.4
a n = 2; b ref. 9
Experimental
derivative 22f showed a surprising 2-fold improvement in activity
over the corresponding nitroso derivative 19d. Improved potency
was also observed for the racemic sec-butoxy derivative 22h, which
displayed potent activity (CDK2; IC50 = 3.8 nM). The enantiomers
of 22h were prepared from the respective chiral sec-butanols, and
the R-enantiomer 22j showed sub-nanomolar potency against
CDK2. By contrast, the 4-cyclohexylmethoxy and the rac-1-
cyclohexylethoxy derivatives (22k and 22l) were 49 and 92-fold
less active than 19a, respectively. The 4-cyclohexylmethoxy-5-
methyliminyl derivatives, 23a and 23b, were significantly less
potent than the corresponding 5-nitroso derivatives (19a and 19b).
The racemic sec-butoxy-5-methylimine 23c, however, retained
potency but was less active than the corresponding 5-formyl
derivative 22h.
Reagents were purchased from fine chemicals vendors, and used as
received unless otherwise stated. Solvents were purified and stored
according to standard procedures. Petrol refers to that fraction in
the boiling range 40–60 ◦C. Melting points were obtained on a
Stuart Scientific SMP3 apparatus and are uncorrected. Thin layer
chromatography was performed using silica gel plates (Kieselgel
60F254; 0.2 mm), and visualized with UV light or potassium
permanganate. Chromatography was conducted under medium
pressure in glass columns or using a Biotage SP4 instrument
in prepacked columns (FLASH+ Silica columns (40–63 mm,
˚
˚
60 A), NH FLASH+ cartridges (40–75 mm, 100 A) and C18
˚
FLASH+ cartridges (40–63 mm, 90 A)). Semi-preparative HPLC
was conducted using a Varian Prostar instrument equipped with
Three compounds were selected for profiling in a panel of
CDKs (Table 4). The 5-nitrososulfonamide 19a showed 100-
fold selectivity for CDK2 over CDK1/cyclinB, 740-fold selec-
tivity against CDK9/cyclinT, and > 1000-fold selectivity vs.
CDK4/cyclinD and CDK7/cyclinH. Interestingly, the (R)-sec-
a Waters XTerra column (RP18 OBD 10 mm, 150 ¥ 30 mm) or
˚
Phenomenex Synergi 4u Fusion-RP (80 A, 250 ¥ 21.2 mm) as
indicated, monitoring by UV at l = 270 nm. Proton (1H) and
carbon (13C) nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker Spectrospin AC 300E (1H at 300 MHz, 13
C
Table 4 CDK selectivity and cell growth inhibition for compounds 6189, 6353, and 6407
IC50/nM
Compound
CDK1/B
CDK2/A
CDK4/D
CDK7/H
CDK9/T
GI50/mMa
19a
22h
22j
100 10
73
9
1
3.8
0.77
0.3
1500 500
3200
1400
2800 1300
890
710
740 100
87
51
2.1
0.93
0.57
a Concentration required to inhibit the growth of SKUT1B tumour cells by 50%
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at 75 MHz) or a Jeol JNM-LA500 spectrometer (1H at 500 MHz,
13C at 125 MHz) employing the solvent as internal standard.
Coupling constants J values are given in Hz. NH signals appeared
as broad singlets (br s) exchangeable with D2O. Mass spectra
were determined on a Micromass Autospec M spectrometer
in electron impact (EI) mode. Liquid Chromatography-Mass
Spectrometry (LCMS) was carried out on a Micromass Platform
instrument operating in positive and negative ion electrospray
mode, employing either a Waters Symmetry (30 ¥ 4.6 mm; C18;
5 min run) or Waters Atlantis (50 ¥ 4.6 mm; C18; 12 min
run) column with 0.05% aqueous formic acid and acetonitrile
(5 to 95% organic). Elemental combustion analyses were either
recorded on Carlo-Erba instrument 1106 analyser, in-house, or
were performed either by Medac Ltd (Brunel Science Centre,
Egham, Surrey, TW20 0JZ), or by the School of Pharmacy at
London University (29–39 Brunswick Square, London, WC1N
1AX). Accurate mass analyses were measured using a Finnigan
MAR 95 XP or a Finnigan MAR 900 XLT at Swansea EPSRC
National Mass Spectrometry Service Centre (EPSRC, Chemistry
Department, University of Wales Swansea, Wales, SA2 8PP). Final
compounds were found to be >97% purity by LCMS and NMR
analysis or gave satisfactory CNH analysis. Experimental details
for additional compounds are found in the ESI.†
for 1 h, then allowed cool to rt, a white precipitate was formed.
Water (30 mL) was added and the mixture was neutralised with
a 2.5 M NaOH solution, extracted with EtOAc (4 ¥ 40 mL), and
the combined organic layers were washed with water (40 mL),
dried (Na2SO4) and concentrated in vacuo. Recrystallisation
(EtOAc,Petrol) gave 5◦as a white solid (1.92 g, 71%). M.p. 193–
◦
194 C; (Lit 195–196 C). UV lmax (EtOH) 274 and 234 nm; IR
n
max 3471, 3337, 3140, 2923, 2848, 1618, 1559, 1433, 1192 cm-1. dH
(300 MHz, d6-DMSO): 0.96–1.25 (5H, m, C6H11), 1.67–1.76 (6H,
m, C6H11), 3.98 (2H, d, J = 6.0 Hz, OCH2), 6.11 (2H, s, NH2,
ex), 6.28 (2H, s, NH2, ex). dC (75 MHz, d6-DMSO): 25.6 (C6H11),
26.4 (C6H11), 29.4 (C6H11), 37.2 (C6H11), 71.1 (OCH2), 71.9 (C5),
161.4 (C6), 162.0 (C4), 165.1 (C2). MS (ESI+) m/z 301 and 303
[M+H]+,HRMS (ESI+) m/z: Calc. for C11H1779BrN4O: 301.0659
[M+H]+. Found 301.0655 [M+H]+. C11H17BrN4O: requires C,
43.87; H, 5.69; N,18.59; found: C, 43.98; H, 5.72; N, 18.55%.
N,N¢-(5-Bromo-6-cyclohexylmethoxypyrimidine-2,4-
diyl)diacetamide (8)
5-Bromo-6-cyclohexylmethoxypyrimidine-2,4-diamine (5) (2.00 g,
6.64 mmol) in a mixture of acetic anhydride (16 mL) and acetic acid
(16 mL) was heated at reflux for 18 h. The mixture was cooled to rt,
poured into ice and water, basified with conc. NH3, and extracted
with EtOAc (4 ¥ 40 mL). The combined organic layers were washed
with aq. sodium bicarbonate (sat; 30 mL), water (30 mL), dried
(Na2SO4) and then concentrated in vacuo giving 8 as an off-white
solid (2.35 g, 95%). M.p.: 179–182 ◦C. UV lmax (EtOH): 253, 276
and 299 nm; IR umax 3391, 3253, 2921, 2853, 1716, 1672, 1577,
1508, 1424, 1190 cm-1; dH (300 MHz, d6-DMSO) 1.02–1.23 (5H,
m, C6H11), 1.69–1.75 (6H, m, C6H11), 2.17 (3H, s, COCH3), 2.20
(3H, s, COCH3), 4.19 (2H, d, J = 6.1 Hz, OCH2), 9.96 (1H, s,
NH, ex), 10.5 (1H, s, NH, ex); dC (75 MHz, d6-DMSO) 24.1, 25.2,
25.5, 26.3, 29.3, 36.9, 72.9, 90.5, 155.1, 157.5, 166.7, 169.3, 169.6.
MS (ESI+) m/z 385 and 387 [M+H]+; HRMS (ESI+) m/z: Calc.
for C15H2179BrN4O3 385.0870 [M+H]+. Found 385.0873 [M+H]+.
C15H21BrN4O3: requires C, 46.76; H, 5.49; N, 14.54%. Found C,
46.52; H, 5.79; N, 14.23%.
4-Chloro-5-nitropyrimidine-2,6-diamine (13)
4-Chloro-2,6-diaminopyrimidine (0.5 g, 3.5 mmol) was added to a
mixture of fuming HNO3 (0.5 mL) and conc. H2SO4 (2.5 mL).
The resulting mixture was stirred at 35 ◦C for 30 min, then
poured slowly onto crushed ice with vigorous stirring yielding a
yellow solid. The mixture was adjusted to pH 8–9 by the dropwise
◦
addition of conc. aq. NH3, at -10 C, then filtered, washed with
copious amounts of water and dried in vacuo over P2O5 (0.28 g,
◦
42%): m.p. 218–220 ◦C; (Lit. 220–222 C);14 dH (300 MHz d6-
DMSO) 7.46 (1H, s, NH2, ex), 7.63 (1H, s, NH2, ex), 8.13 (2H, s
br, NH2, ex). MS (ESI+) m/z 190 [M+H]+.
4-Cyclohexylmethoxy-5-nitropyrimidine-2,6-diamine (14)
Compound 13 (0.11 g, 0.58 mmol) was added to a mixture of
sodium tert-butoxide (0.07 g, 0.70 mmol), in tert-butanol (10 mL)
and cyclohexylmethanol (0.11 mL, 0.87 mmol) and stirred at
30 ◦C for 22 h, then concentrated in vacuo yielding a yellow solid.
Chromatography (silica; 40% EtOAc, petrol), gave 14 as a yellow
solid (0.04 g, 25%): m.p. 177–178 ◦C. UV lmax = 328 and 211 nm,
IR nmax 1340, 1544, 1618, 2850–2921, 3152, 3360–3481 cm-1. dH
(300 MHz d6-DMSO) 1.00–1.26 (5H, m, C6H11), 1.68–1.80 (6H,
m, C6H11), 4.10 (2H, d, J = 6.0 Hz, CH2O), 7.20 (2H, s, NH2, ex),
7.89 (2H, s, NH2, ex) ppm; dC (75 MHz d6-DMSO) 25.6 (C6H11),
26.3 (C6H11), 29.4 (C6H11), 37.0 (C6H11), 72.2 (OCH2), 83.0 (C5),
160.3 (C6), 161.2 (C4), 165.0 (C2). MS (ESI+) m/z 268 [M+H]+;
C11H17N5O3·0.2 EtOAcrequires:C, 49.75;H, 6.58;N, 24.58; found:
C, 49.58; H, 6.38; N, 24.43.
2,4-Diamino-5-cyano-6-cyclohexylmethoxypyrimidine (9)13
Compound 8 (0.4 g, 1.04 mmol) and CuCN (0.112 g, 1.25 mmol)
were heated at reflux in DMF (8.0 mL) for 5 h. Ethane-1,2-diamine
(20 mL) was added and the reaction mixture was stirred for further
2 h. The mixture was filtered through Celite and the filter pad was
washed with DMF (20 mL). The filtrate was extracted with EtOAc
(60 mL) and washed with saturated aq. sodium chloride (30 mL).
The organic layer was dried (Na2SO4) and concentrated in vacuo.
Chromatography (silica; 20–80% EtOAc, petrol) gave 9 as a pale
◦
yellow solid (0.212 g, 82%). M.p.: 186–188 C. UV lmax (EtOH):
252 and 266 nm. IR umax 3317, 3233, 2919, 2848, 2208, 1607, 1573,
1547, 1348, 1232 cm-1. dH (300 MHz, d6-DMSO): 0.96–1.26 (5H,
m, C6H11), 1.64–1.75 (6H, m, C6H11), 4.06 (2H, d, J = 6.0 Hz,
OCH2), 6.88 (2H, s, NH2 ex), 6.93 (2H, s, NH2 ex); dC (75 MHz,
d6-DMSO) 25.5, 26.3, 29.3, 37.0, 63.1, 71.1, 116.4, 163.3, 166.0,
171.4; MS (ESI+) m/z 248 [M+H]+; HRMS (ESI+) m/z: Calc. for
C12H17N5O: 248.1506 [M+H]+. Found 248.1504 [M+H]+.
5-Bromo-6-cyclohexylmethoxypyrimidine-2,4-diamine (5)10
To 6-cyclohexylmethoxypyrimidine-2,4-diamine (3f) (2.00 g,
9.01 mmol) in AcOH (10.0 mL) was added NBS (1.60 g,
9.01 mmol) slowly, in small portions. The temperature was
◦
increased to 60 C and the reaction mixture was allowed to stir
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2,4-Diamino-6-cyclohexylmethoxy-5-formylpyrimidine (12)13,20
solution of 2,4-diamino-5-cyano-6-cyclohexylmethoxy-
ex), 5.38 (1H, br, NH ex), 9.27 (1H, br, NH ex); dC (75 MHz,
CDCl3): 23.9, 24.6, 28.2, 31.2, 35.6, 70.4, 91.8, 160.3, 164.8, 169.4,
195.1. MS (ESI+) m/z 265.28 [M+H]+; HRMS (ESI+) m/z: Calc.
for C13H20N4O2: 265.1659 [M+H]+. Found 265.1656 [M+H]+.
A
pyrimidine (9) (0.20 g, 0.81 mmol) in water (7.2 mL) and sulfuric
acid (1.2 mL) was stirred with 5% palladium on charcoal (0.094 g)
under H2 (1 atm) at rt for 48 h. The mixture was filtered through
Celite and the filter pad was washed with hot water (200 mL) and
hot methanol (200 mL). The filtrate was neutralised (NaOH, 2M),
concentrated in vacuo, and the residues extracted with EtOAc (4 ¥
50 mL). The combined organic layers were washed with water
(20 mL) and dried (Na2SO4) and concentrated in vacuo giving
white solid. Chromatography (Biotage C18; 50-25% H2O, MeOH,
1% formic acid) and recrystallisation (methanol) gave 12 as a
white solid (0.151 g, 75%). M.p.: 160–163 ◦C; (Lit: 118–120 ◦C).20
UV lmax (EtOH): 291.6 nm; IR nmax 3327, 3194, 3147, 2916, 2846,
1662, 1586, 1528, 1342, 1259 cm-1; dH (300 MHz, d6-DMSO)
0.92–1.35 (5H, m, C6H11), 1.74–1.82 (6H, m, C6H11), 4.16 (2H,
d, J = 6.0 Hz, OCH2), 7.15 (2H, s, NH2 ex), 7.45 (1H, s, NH2
ex), 8.36 (1H, s, NH2 ex), 9.89 (1H, s, CHO) ppm; dC (75 MHz,
d6-DMSO) 25.2, 25.9, 29.1, 36.7, 70.5, 91.7, 163.8, 164.4, 171.6,
184.4 ppm. MS (ESI+) m/z 251.02 [M+H]+. HRMS (ESI+)
m/z: Calc. for C12H18N4O2: 251.1503 [M+H]+. Found 251.1506
[M+H]+.
5-Iodo-6-(cyclohexylmethoxy)pyrimidine-2,4-diamine. (6)
To a solution of 6-cyclohexylmethoxypyrimidine-2,4-diamine (3f)
(0.20 g, 0.90 mmol, 1 eq.) in AcOH (2.0 mL) was added NIS
(0.24 g, 1.08 mmol). The resulting mixture was stirred for 1 h at
◦
60 C, then allowed to cool to rt, water (5 mL) was added, then
neutralised NaOH (2.5 M), and extracted with DCM (3 ¥ 30 mL).
The combined organic layers were washed with water (30 mL) and
dried (Na2SO4) and concentrated in vacuo giving a solid residue.
Recrystallisation (EtOAc, Petrol) gave 6 as an off-white solid
(0.23 g, 73%) m.p.: 177–178 ◦C. UV lmax (EtOH): 325 and 228 nm;
IR nmax 3477, 3332, 3214, 2914, 2848, 1619, 1582, 1541, 1423,
1167 cm-1; dH (300 MHz, d6-DMSO): 0.95–1.25 (5H, m, C6H11),
1.61–1.78 (6H, m, C6H11), 3.96 (2H, d, J = 6.4 Hz, OCH2), 6.08
(4H, s, NH2 ex); dC (75 MHz, d6-DMSO): 25.6, 26.4, 29.5, 37.3,
43.8, 71.4, 162.9, 164.7, 167.9. MS (ESI+) m/z 349.12 [M+H]+;
HRMS (ESI+) m/z: Calc. for C11H17IN4O: 349.0522 [M+H]+.
Found 349.0522 [M+H]+. C11H17IN4O.0.2 CHCl3: requires C,
59.51; H, 6.71; N, 18.24%; found C, 59.18; H, 6.47, N, 17.96%.
6-Cyclohexylmethoxy-5-(1-iminoethyl)pyrimidine-2,4-diamine
(10)12
4-Amino-2,6-dichloro-5-formylpyrimidine (20)12
To a solution of 9 (0.50 g, 2.02 mmol) in THF (50 mL), under N2,
in an ice bath was added dropwise methyl magnesium bromide
(3.0 M in THF, 3.37 mL, 10.1 mmol), over 10 min while stirring
at 0 ◦C. The mixture was stirred 2 h at 0 ◦C, then heated at reflux
for 24 h. Further methyl magnesium bromide (3.0 M in THF,
6.74 mL, 20.2 mmol) was added over the next 48 h with continued
heating, until the reaction came to completion. The mixture was
cooled to rt, quenched with aq. ammonium chloride (sat, 30 mL),
and extracted with EtOAc (3 ¥ 30 mL). The combined organic
layers were washed with water (40 mL), dried (Na2SO4) and
concentrated in vacuo yielding a yellow powder. Recrystallisation
(EtOAc, petrol) gave 10 as yellow solid (2.35 g, 95%), m.p.: 141–
143 ◦C. UV lmax (EtOH): 240, 312 nm; IR nmax 3365, 3210, 3127,
2984, 2914, 1655, 1554, 1418, 1253 cm-1; dH (300 MHz, d4-MeOD):
0.99–1.32 (5H, m, C6H11), 1.71–180 (6H, m, C6H11), 2.45 (3H, s,
CH3), 4.08 (2H, d, J = 6.0 Hz, OCH2); dC (75 MHz, CDCl3)
26.1, 26.8, 30.4, 33.1, 37.9, 72.1, 91.4, 161.4, 165.8, 170.2, 175.3.
MS (ESI+) m/z 264.25 [M+H]+. HRMS (ESI+) m/z: Calc. for
C13H21N5O: 264.1819 [M+H]+. Found 264.1821 [M+H]+.
Dry N,N-dimethylformamide (7 mL) was added slowly with stir-
ring over 15 min to phosphorous oxychloride (22 mL, 0.24 mol),
at ~ 5 ◦C. The reaction mixture was warmed gently to dissolve the
resulting precipitate, and 4-amino-2,6-dihydroxypyrimidine (5.6 g,
0.044 mmol) was added in small portions over 10 min, then heated
for 5 h at 105 ◦C, producing a dark red–brown solution. Remaining
phosphorous oxychloride was removed by distillation in vacuo,
leaving a viscous dark brown oil, which was mixed with crushed ice
and allowed to stand at rt overnight giving a red–orange solution
and a dark yellow solid. The solid was collected by filtration and
the filtrate was treated with ammonium hydroxide in portions to
pH ~ 7 and left to stand overnight. The yellow–brown precipitate,
was collected by filtration. The combined precipitates were dried in
vacuo over P2O5 overnight and then extracted with hot EtOAc (6 ¥
200ml)leavingabrowninsolubleresiduewhichwasdiscarded. The
solvent was removed in vacuo to give a bright yellow solid, which
after recrystallisation (EtOAc) gave 20 (5.3 g, 63%) m.p.: 187–
188 ◦C. UV lmax (EtOH): 250 and 316 nm. IR nmax 3354, 3188, 2877,
1728, 1630, 1521 cm-1. dH (300 MHz, d6-DMSO): 8.74 (1H, s, NH
exchangeable with D2O), 9.11 (1H, s, NH exchangeable with D2O),
10.18 (1H, s, CHO). dC (75 MHz, d6-DMSO): 90.6, 149.6, 157.9,
165.4, 186.7. MS (ESI+) m/z 191.92 [M+H]+. HRMS (EI) m/z:
Calc. for C5H3Cl2N3O, 190.9659. Found 190.9661. C5H3Cl2N3O
requires C, 31.28; H, 1.57; N, 21.89%. Found C, 31.50; H, 1.58, N,
21.84%.
1-(2,4-Diamino-6-cyclohexylmethoxypyrimidin-5-yl)ethanone (11)
A solution of HCl (2M; 10 mL) was added dropwise to a solution
of 10 (0.075 g, 0.285 mmol) in THF (10 mL). The resulting mixture
was stirred at rt for 16 h, then neutralised by dropwise addition
of NaOH (2M), and extracted with EtOAc (3 ¥ 30 mL). The
combined organic layers were washed with water (30 mL) and
dried (Na2SO4) and concentrated in vacuo giving a yellow powder.
Recrystallisation (EtOAc, petrol), gave 11 as yellow solid (0.063 g,
General procedure A
Sodium (1.5 eq.) was added to the appropriate alcohol (20 mL) and
the resulting mixture was stirred and heated for 2 h, then 4-chloro-
2,6-diaminopyrimidine (1 eq.) was added and stirring continued
5 h with heating to the specified temperature. After cooling, the
mixture was neutralised with glacial acetic acid and concentrated
◦
84%), m.p. 149–151 C. UV lmax (EtOH): 283 nm. IR nmax 3340,
3221, 2923, 2849, 1683, 1548, 1524, 1431, 1244 cm-1; dH (300 MHz,
CDCl3): 0.93–1.28 (5H, m, C6H11), 1.62–1.77 (6H, m, C6H11), 2.47
(3H, s, CH3), 4.08 (2H, d, J = 6.0 Hz, OCH2), 4.93 (2H, br, NH2
2404 | Org. Biomol. Chem., 2010, 8, 2397–2407
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in vacuo to yield a yellow oil, which was diluted with water (25 mL)
and extracted with EtOAc (4 ¥ 25 mL). The combined organic
layers were dried (MgSO4) and concentrated in vacuo giving the
desired product.
(1.5 eq.) was added dropwise. The resulting mixture was allowed
to stir at rt for 72 h then concentrated in vacuo to yield a yellow
oil, which was triturated with Et2O in an ice-bath at 0 ◦C,
giving a white precipitate which was removed by filtration. The
filtrate was concentrated in vacuo, water (30 mL) was added
and the mixture was extracted with EtOAc (3 ¥ 30 mL). The
combined organic layers were dried (Na2SO4) and concentrated
in vacuo. Chromatography (silica; 20–80% EtOAc, petrol) and
recrystallisation (MeOH) gave the desired product 16a–c.
6-Methoxypyrimidine-2,4-diamine (3a)
General procedure A. Sodium (0.38 g, 50 mmol), methanol
(50 mL), 4-chloro-2,6-diaminopyrimidine (5.00 g, 34.6 mmol), at
reflux for 1 h. After cooling, the mixture was neutralised with
glacial acetic acid and concentrated in vacuo to yield a yellow oil,
which was diluted with water (25 mL) and extracted with EtOAc
(4 ¥ 25 mL). The combined organic layers were dried (NaSO4)
and concentrated in vacuo giving 3a as a white solid (4.64 g, 96%).
m.p. 165.9–167.8 ◦C. lmax (CH3OH) 264.5 nm; IR nmax/cm-1: 3485,
3322, 3142, 1391, 1620, 1568, 1445. dH (300 MHz, d6-DMSO) d =
3.67 (3H, s, CH3), 5.04 (1H, s, H5), 5.90 (2H, br s, D2O exch, NH2),
6.02 (2H, br s, D2O exch, NH2); dC (75 MHz, d6-DMSO) d = 52.7,
76.6, 163.4, 166.4, 171.0; HRMS (ESI+) m/z: Calc. for C5H8N4O:
141.0771 [M+H]+. Found 141.0770 [M+H]+.
2-Butylsulfanyl-6-cyclohexylmethoxypyrimidin-4-amine (16a)
General procedure C. 15 (9.00 g, 45.2 mmol), cyclohexyl-
methanol (8.34 mL, 67.8 mmol), triphenylphosphine (17.76 g,
67.8 mmol), diethyl azodicarboxylate (10.7 mL, 67.8 mmol) gave
16a as a white solid (10.1 g, 76%) m.p.: 81–83 ◦C. UV lmax (EtOH):
257 nm; IR nmax 3438, 3283, 3167, 2961, 2929, 1631, 1573, 1542,
1347, 1243, 1019 cm-1. dH (300 MHz, CDCl3): 0.93 (3H, t, J =
7.3 Hz, CH3CH2CH2CH2S), 1.02–1.32 (7H, m, 5H C6H11 + 2H
CH3CH2CH2CH2S), 1.38–1.51 (2H, m, CH3CH2CH2CH2S), 1.65–
1.80 (6H, m, C6H11), 3.06 (2H, t, J = 7.1 Hz, CH3CH2CH2CH2S),
4.07 (2H, d, J = 6.3 Hz, OCH2), 4.75 (2H, br, NH2 ex), 5.44
(1H, s, H-5); dC (75 MHz, CDCl3): 13.9, 22.4, 26.1, 26.8, 30.2, 30.8,
32.2, 37.9, 71.7, 83.4, 164.5, 170.5, 171.3. MS (ESI+) m/z 296.35
[M+H]+; HRMS (ESI+) m/z: Calc. for C15H25N3OS: 296.1791
[M+H]+. Found 296.1792 [M+H]+.
General
procedure
B.
A
mixture
of
5-bromo-6-
cyclohexylmethoxypyrimidine-2,4-diamine (5) (0.6 g, 1.99 mmol,
1.0 eq.), the appropriate aryl or heteroaryl boronic acid
(2.39 mmol, 1.2 eq.), tetrakis(triphenylphosphine)palladium(0)
(0.115 g, 0.1 mmol, 5% mol eq.), K2CO3 solution (4 M, 2 mL,
8 mmol, 4.0 eq.), and 1,2-dimethoxyethane (10 mL) was heated
◦
under microwave irradiation at 170 C, for 1–2 h. The resulting
General procedure D. To a solution of the appropriate 2-(n-
butylsulfanyl)-6-alkoxyoxypyrimidin-4-amine (16a–c) (1 eq.) in
DCM under N2, m-CPBA (2.5–3 eq.) was added slowly. The
resulting mixture was stirred at rt for 24 h, then concentrated
in vacuo. The residue was extracted with EtOAc (3 ¥ 30 mL). The
combined organic fractions were washed with aq. sodium sulfite
(sat.; 30 mL), aq. sodium bicarbonate (sat.; 2 ¥ 30 mL) and water
(2 ¥ 30 mL), dried (Na2SO4) and concentrated in vacuo to give a
white solid. Chromatography (silica; 20–80% EtOAc, petrol) gave
17a–c as a white solid.
mixture was filtered through Celite, washed successively with
MeOH (4 ¥ 30 mL) and DCM (4 ¥ 30 mL). The combined fractions
were concentrated in vacuo. To remove further palladium residues,
a thiol-functionalized resin PL-Thiol SPE Tube (StratospheresTM
SPE) was employed. The SPE tube was preconditioned with
methanol, then the compound sample, previously dissolved in the
smallest amount possible of methanol, was loaded and allowed
to pass through the SPE media under gravity and the filtrate
concentrated in vacuo. The residues were dissolved in water
(30 mL) and extracted with EtOAc (3 ¥ 30 mL). The combined
organic fractions were dried (Na2SO4) and concentrated in vacuo.
Chromatography (silica; EtOAc, petrol) followed by HPLC,
Waters XTerra column, gave the desired product 7a–c as a white
solid.
2-Butylsulfonyl-6-(cyclohexylmethoxy)pyrimidin-4-amine (17a)
General procedure D. 16a (8.0 g, 27.1 mmol), DCM (150 mL),
m-CPBA (18.7 g, 0.11 mol). Recrystallisation (EtOAc, petrol)
gave 17a as a white solid (7.18 g, 81%), m.p.: 142–144 ◦C. UV
lmax (EtOH): 279 nm; IR nmax/cm-1: 3416, 3323, 3121, 2962,
2927, 2880, 1636, 1595, 1533, 1460, 1379, 1231; dH (300 MHz,
CDCl3): 0.93 (3H, t, J = 7.3 Hz, CH3CH2CH2CH2SO2), 1.01–1.27
(7H, m, 12H C6H11 and CH3CH2CH2CH2SO2), 1.39–1.52 (2H, m,
CH3CH2CH2CH2SO2), 1.71–1.83 (2H, m, C6H11), 3.39 (2H, t, J =
7.8 Hz, CH3CH2CH2CH2SO2), 4.08 (2H, d, J = 6.1 Hz, OCH2),
5.80 (2H, br, NH2, ex), 5.90 (1H, s, H-5). dC (75 MHz, CDCl3):
13.7, 22.1, 24.5, 26.0, 26.7, 30.1, 37.8, 51.2, 72.9, 89.1, 165.2, 165.6,
171.4; MS (ESI+) m/z 328.36 [M+H]+; HRMS (ESI+) m/z: Calc.
for C15H25N3O3S: 328.1689 [M+H]+. Found 328.1684 [M+H]+.
6-Cyclohexylmethoxy-5-phenylpyrimidine-2,4-diamine (7a)
General procedure B. Phenyl boronic acid (0.73 g, 6.0 mmol,
3 eq.). Chromatography and HPLC, Waters XTerra column, gave
7a as an off-white solid (0.126 g, 23%), m.p.: 103–105 ◦C. UV lmax
(EtOH): 271 and 206 nm; IR nmax 3311, 3170, 3057, 2919, 2856,
1611, 1554 cm-1; dH (300 MHz, CDCl3): 0.77–1.18 (6H, m, C6H11),
1.58–1.78(5H, m, C6H11), 3.93 (2H, d, J = 6.0 Hz, OCH2), 4.48
(2H, s, NH2 ex), 4.62 (2H, s, NH2 ex), 7.21–7.35 (5H, m, ArH); dC
(75 MHz, CDCl3) 26.1, 26.9, 30.1, 37.8, 71.4, 94.2, 127.1, 128.8,
131.1, 133.9, 161.6, 162.9, 167.9; MS (ESI+) m/z 299 [M+H]+.
C17H22N4O: requires C, 68.43; H, 7.43; N,18.78%. Found C, 68.53;
H, 7.45; N, 18.79%.
General procedure E. A solution of the required pyrimidine
(0.50 g, 2.62 mmol, 1 eq.) and the chosen aniline (5.76 mmol, 2.2
eq.) in trifluoroethanol (10 mL) with trifluoroacetic acid (1.0 mL,
13.1 mmol, 5 eq.) was stirred for 48 h at rt or heated at reflux 24 h
as specified. The solvents were removed in vacuo, water (30 mL)
was added and the pH was adjusted to neutral with saturated aq.
sodium bicarbonate and the mixture was extracted with EtOAc
General procedure C.
A
mixture of 6-amino-2-(n-
butylsulfanyl)-4(3H)-pyrimidinone (15) (1 eq.), the appropriate
alcohol (1.5 eq.), and triphenylphosphine (1.5 eq.) in THF (anh)
was stirred at 5 ◦C in an ice-bath and diethyl azodicarboxylate
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(3 ¥ 30 mL). The combined organic layers were washed with water
(2 ¥ 30 mL) and dried (Na2SO4), and the solvent was removed in
vacuo. The compounds were purified as specified.
rac-4-(4-Amino-6-sec-butoxy-5-formylpyrimidin-2-
ylamino)benzenesulfonamide (22h)
General procedure F. 4-(4-amino-6-chloro-5-formylpyrimidin-
2-ylamino)benzenesulfonamide (21a) (0.40 g, 1.22 mmol), sec-
butanol (2.3 mL, 24.5 mmol). Chromatography (silica; 2–15%
MeOH, DCM) and HPLC, Phenomenex Synergi (35–100%
MeCN, water) gave 22h as a white solid (97 mg, 11%), m.p. 265–
267 ◦C. UV lmax (EtOH): 325 nm; IR nmax 3278, 3215, 2923, 2852,
1645, 1564, 1321, 1149 cm-1; dH (300 MHz, d4-MeOD) 1.00 (3H,
t, J = 7.5 Hz, CH3CHORCH2CH3), 1.38 (3H, d, J = 6.2 Hz,
CH3CHORCH2CH3), 1.65–1.86 (2H, m, CH3CHORCH2CH3),
5.30–5.41 (1H, m, CH3CHORCH2CH3), 7.82 (2H, d, J = 8.9 Hz,
ArH(2,6)), 7.95 (2H, d, J = 8.9 Hz, ArH(3,5)), 9.97 (1H, s, CHO);
dC (75 MHz, d4-MeOD): 10.1, 19.8, 30.6, 75.9, 96.1, 120.6, 128.0,
138.1, 144.7, 156.7, 162.1, 170.4, 188.0. MS (ESI+) m/z 366.31
[M+H]+; HRMS (ESI+) m/z: Calc. for C15H19N5O4S: 366.1231
[M+H]+. Found 366.1237 [M+H]+.
4-(4-Amino-6-chloro-5-formylpyrimidin-2-
ylamino)benzenesulfonamide (21a)
General
procedure
E. 4-amino-2,6-dichloro-5-formyl-
pyrimidine (20) (0.5 g, 2.62 mmol), sulfanilamide (0.54 g,
3.13 mmol), trifluoroacetic acid (1.0 mL, 13.1 mmol) and
trifluoroethanol (10 mL). Recrystallisation (methanol) gave 21a
as a yellow solid (0.63 g, 74%) m.p.: 244 –246 ◦C. UV lmax (EtOH):
332 and 262 nm. IR nmax/cm-1: 3325, 3194, 3155, 2852, 1713,
1574, 1503, 1342, 1163, 1061; dH (300 MHz, d6-DMSO): 7.28
(2H, s, NH2 exchangeable with D2O), 7.74 (2H, d, J = 8.2 Hz,
ArH(2,6)), 8.01 (2H, d, J = 8.2 Hz, ArH(3,5)), 8.51 (1H, br,
NH exchangeable with D2O), 8.64 (1H, br, NH exchangeable
with D2O), 10.04 (1H, s, CHO), 10.58 (1H, br, NH exchangeable
with D2O). dC (75 MHz, d6-DMSO): 102.8, 120.2, 126.7, 138.6,
142.4, 159.0, 163.7, 165.0, 188.3. MS (ESI+) m/z 328.10 [M+H]+.
HRMS (ESI+) m/z: Calc for C11H10ClN5O3S: 328.0266 [M+H]+.
Found 328.0264 [M+H]+. C11H10ClN5O3S·0.1CH2Cl2: requires C,
39.65; H, 3.06; N, 20.83%. Found C, 39.58; H, 2.98; N, 20.95%.
(S)-4-(4-Amino-6-sec-butoxy-5-formylpyrimidin-2-
ylamino)benzenesulfonamide (22j)
General procedure F. 4-(4-amino-6-chloro-5-formylpyrimidin-
2-ylamino)benzenesulfonamide (21a) (0.40 g, 1.22 mmol), (S)-
sec-butanol (1.1 mL, 12.2 mmol). Chromatography (silica; 2–
15% MeOH–DCM) and HPLC, Phenomenex Synergi (35–100%
MeCN, water), gave 22j as a white solid (31 mg, 7%), m.p.: 269–
270 ◦C. UV lmax (EtOH): 319 nm; IR nmax 3275, 3230, 2959, 2924,
2861, 1650, 1558, 1377, 1156 cm-1; dH (300 MHz, d3-MeCN) 0.74
(3H, t, J = 7.4 Hz, CH3CHORCH2CH3), 1.13 (3H, d, J = 6.2 Hz,
CH3CHORCH2CH3), 1.39–1.61 (2H, m, CH3CHORCH2CH3),
5.01–5.09 (1H, m, CH3CHORCH2CH3), 5.36 (2H, s, NH2), 6.06
(1H, br, NH), 7.56 (2H, d, J = 8.9 Hz, ArH(2,6)), 7.68 (2H, d,
J = 8.9 Hz, ArH(3,5)), 7.97 (1H, br, NH), 8.34 (1H, s, NH), 9.80
(1H, s, CHO); dC (75 MHz, d4-MeOD) 10.2, 20.0, 30.4, 76.4, 90.4,
121.3, 128.3, 137.0, 144.7, 155.9, 162.5, 173.2, 188.4. MS (ESI+)
m/z 366.24 [M+H]+. HRMS (ESI+) m/z: Calc. for C15H19N5O4S:
366.1231 [M+H]+. Found 366.1236 [M+H]+.
General procedure F. The chosen alcohol (10 eq.) was added
dropwise to a suspension of sodium hydride (5 eq.) in THF (anh;
5 mL), and the mixture was stirred at rt under N2 for 30 min.
The chosen 4-amino-6-chloro-2-(arylamino)-5-formylpyrimidine
(1 eq.) was added slowly, in small portions and the reaction mixture
was heated at reflux 16 h, then allowed to cool to rt, quenched
with water (10 mL), and acidified (2 M HCl) to pH < 2, then THF
(10 mL) was added, the resulting mixture was stirred overnight,
neutralised (saturated aq. sodium bicarbonate), and concentrated
in vacuo. The residue was extracted with EtOAc (4 ¥ 30 mL) and the
combined organic layers were dried (Na2SO4), and concentrated
in vacuo to give a yellow–orange oil. Chromatography (silica; 40–
100% EtOAc, petrol, or 10% MeOH, DCM) and recrystallisation
(methanol) or HPLC purification, gave the product as a white
solid.
General procedure G. The appropriate O4-substituted pyrim-
idine (1 eq.) was dissolved in 30% aq. acetic acid (1 ml–4 ml) at
rt. The temperature was increased to 80 ◦C and sodium nitrite
(1.3 eq.) in water (1 ml–3 ml) was added dropwise to give a
purple precipitate. The reaction was maintained at 80 ◦C for
40 min, allowed to cool to rt and the precipitate was collected
by filtration. The crude product was purified by recrystallisation
from the appropriate solvent.
4-Amino-6-cyclohexylmethoxy-2-(4-methoxyphenylamino)-5-
formylpyrimidine (22a)
General
procedure
F. 4-amino-6-chloro-2-(4-methoxy-
phenylamino)-5-formylpyrimidine (21b) (0.20 g, 0.72 mmol),
cyclohexylmethanol (0.88 mL, 7.2 mmol). Chromatography
(silica; 40–100% EtOAc, Petrol) and recrystallisation (methanol)
gave 22a as an off-white solid (0.159 g, 62%) m.p. 94–97 ◦C.
UV lmax (EtOH): 325 nm. IR nmax/cm-1: 3311, 3070, 2920, 1663,
1550, 1491, 1236. dH (300 MHz, d4-MeOD): 0.81–1.32 (5H, m,
C6H11), 1.64–1.78 (6H, m, C6H11), 3.72 (3H, s, OCH3), 4.15 (2H,
d, J = 6.0 Hz, OCH2), 6.81 (2H, d, J = 8.9 Hz, ArH(3,5)), 7.48
(2H, d, J = 8.9 Hz, ArH(2,6)), 9.83 (1H, s, CHO); dC (75 MHz,
d4-MeOD): 26.8, 27.6, 30.9, 38.8, 56.1, 72.9, 94.3, 115.1, 124.3,
133.6, 157.8, 162.7, 166.3, 173.6, 187.3. MS (ESI+) m/z 357.33
[M+H]+. HRMS (ESI+) m/z: Calc. for C19H24N4O3: 357.1921
[M+H]+. Found 357.1922 [M+H]+. C19H24N4O3.0.4 M MeOH
requires C, 63.02; H, 6.96; N, 15.23%; found C, 63.49; H, 6.46, N,
14.78%.
6-Methoxy-5-nitrosopyrimidine-2,4-diamine (4a)
General procedure G. 6-methoxypyrimidine-2,6-diamine
(3a)(0.25 g, 1.78 mmol), 30% aq. acetic acid (5 mL), sodium
nitrite (160 mg, 2.32 mmol) in water (2 mL). The purple precipitate
was filtered off and dried (P2O5) giving 4a. (246 mg, 82%). m.p.
249.2–251.8 ◦C; UV (EtOH): lmax1 = 330 nm; IR nmax 3508, 3282,
1566, 1492, 1441, 1372, 1311 cm-1; dH (300 MHz, d6-DMSO) d
4.05 (3H, s, CH3), 7.81 (1H, br s, NH2), 7.87 (1H, br s, NH2), 8.03
(1H, br s, NH2), 10.07 (1H, br s, NH2); HRMS (ESI+) m/z: Calc.
for C5H7N5O2: 141.0771 [M+H]+. Found 141.0770 [M+H]+.
2406 | Org. Biomol. Chem., 2010, 8, 2397–2407
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General procedure H. To a solution of formylpyrimidine
(22)(0.17 mmol) in EtOH (anh; 3 mL) was added hydroxylamine
hydrochloride (0.014 g, 0.202 mmol), followed by pyridine (anh.,
0.016 mL, 0.202 mmol) and the mixture was stirred 16 h at rt
under N2, then concentrated in vacuo. The residues were diluted
with water (30 mL) and extracted with EtOAc (3 ¥ 40 mL). The
combined organic layers were dried (Na2SO4) and concentrated
in vacuo.
5 H. Hochegger, S. Takeda and T. Hunt, Nat. Rev. Mol. Cell Biol., 2008,
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6 S. Lapenna and A. Giordano, Nat. Rev. Drug Discovery, 2009, 8, 547–
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7 T. G. Davies, J. Bentley, C. E. Arris, F. T. Boyle, N. J. Curtin, J. A.
Endicott, A. E. Gibson, B. T. Golding, R. J. Griffin, I. R. Hardcastle, P.
Jewsbury, L. N. Johnson, V. Mesguiche, D. R. Newell, M. E. M. Noble,
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4-Amino-6-(cyclohexylmethoxy)-2-(4-methoxyphenylamino)-
pyrimidine-5-carbaldehyde oxime (23a)
9 F. Marchetti, K. L. Sayle, J. Bentley, W. Clegg, N. J. Curtin, J. A.
Endicott, B. T. Golding, R. J. Griffin, K. Haggerty, R. W. Harrington,
V. Mesguiche, D. R. Newell, M. E. M. Noble, R. J. Parsons, D. J.
Pratt, L. Z. Wang and I. R. Hardcastle, Org. Biomol. Chem., 2007, 5,
1577–1585.
10 H. I. Skulnick, S. D. Weed, E. E. Eidson, H. E. Renis, D. A. Stringfellow
and W. Wierenga, J. Med. Chem., 1985, 28, 1864–1869.
11 R. J. Griffin and P. R. Lowe, J. Chem. Soc., Perkin Trans. 1, 1992,
1811–1819.
General
procedure
H. 4-amino-6-cyclohexylmethoxy-2-
(4-methoxyphenylamino)-5-formylpyrimidine (22a) (0.06 g,
0.17 mmol). Recrystallisation (EtOAc, petrol) gave 23a as a white
solid (0.047 g, 75%), m.p. 158–160 ◦C. UV lmax (EtOH): 310
and 219 nm. IR nmax 3481, 3343, 3145, 2922, 1580, 1497, 1238,
952 cm-1; dH (300 MHz, CDCl3): 0.96–1.34 (6H, m, C6H11),
1.68–1.82 (5H, m, C6H11), 3.79 (3H, s, OCH3), 4.12 (2H, d, J =
6.1 Hz, OCH2), 6.85 (2H, d, J = 9.0 Hz, ArH(3,5)), 7.07 (1H,
br, NH), 7.44 (2H, d, J = 9.0 Hz, ArH(2,6)), 7.64 (1H, br, NH),
8.47 (1H, s, CHNOH), 10.02 (1H, s, CHNOH); dC (75 MHz,
CDCl3): 26.1, 26.8, 29.9, 37.8, 55.9, 72.1, 85.7, 114.5, 122.3, 133.1,
146.9, 156.2, 159.1, 162.5, 168.9; MS (ESI+) m/z 372.40 [M+H]+;
HRMS (ESI+) m/z: Calc. for C19H25N5O3: 372.2030 [M+H]+.
Found 372.2034 [M+H]+.
12 L. Bell, H. M. McGuire and G. A. Freeman, J. Heterocycl. Chem., 1983,
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13 D. Hockova, A. Holy, M. Masojidkova, G. Andrei, R. Snoeck, E. De
Clercq and J. Balzarini, Bioorg. Med. Chem., 2004, 12, 3197–3202.
14 D. E. O’Brien, C. C. Cheng and W. Peleiderer, J. Med. Chem., 1966, 9,
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15 H. J. Whitfield, R. J. Griffin, I. R. Hardcastle, A. Henderson, J.
Meneyrol, V. Mesguiche, K. L. Sayle and B. T. Golding, Chem.
Commun., 2003, 2802–2803.
16 C. E. Arris, F. T. Boyle, A. H. Calvert, N. J. Curtin, J. A. Endicott,
E. F. Garman, A. E. Gibson, B. T. Golding, S. Grant, R. J. Griffin,
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Acknowledgements
The authors thank BBSRC, EPSRC and Cancer Research UK
for financial support. The use of the EPSRC Mass Spectrometry
Service at the University of Wales (Swansea) is gratefully acknowl-
edged.
17 A. E. Gibson, C. E. Arris, J. Bentley, F. T. Boyle, N. J. Curtin, T. G.
Davies, J. A. Endicott, B. T. Golding, S. Grant, R. J. Griffin, P. Jewsbury,
L. N. Johnson, V. Mesguiche, D. R. Newell, M. E. M. Noble, J. A.
Tucker and H. J. Whitfield, J. Med. Chem., 2002, 45, 3381–3393.
18 V. Mesguiche, R. J. Parsons, C. E. Arris, J. Bentley, F. T. Boyle, N. J.
Curtin, T. G. Davies, J. A. Endicott, A. E. Gibson, B. T. Golding, R. J.
Griffin, P. Jewsbury, L. N. Johnson, D. R. Newell, M. E. M. Noble,
L. Z. Wang and I. R. Hardcastle, Bioorg. Med. Chem. Lett., 2003, 13,
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The Royal Society of Chemistry 2010
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