Synthesis and evaluation of phosphorus containing, specific CDK9/CycT1 inhibitors

Article abstract of DOI:10.1021/jm401742r

Although there is a significant effort in the design of a selective CDK9/CycT1 inhibitor, no compound has been proven to be a specific inhibitor of this kinase so far. The aim of this research was to develop novel and selective phosphorus containing CDK9/CycT1 inhibitors. Molecules bearing phosphonamidate, phosphonate, and phosphinate moieties were synthesized. Prepared compounds were evaluated in an enzymatic CDK9/CycT1 assay. The most potent molecules were tested in cell-based toxicity and HIV proliferation assays. Selectivity of shortlisted compounds against CDKs and other kinases was tested. The best compound was shown to be a highly specific, ATP-competitive inhibitor of CDK9/CycT1 with antiviral activity.

Full text of DOI:10.1021/jm401742r

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Phosphorus Containing, Specific CDK9/
CycT1 Inhibitors
Gabor Nemeth,*,† Zoltan Greff,† Anna Sipos,† Zoltan Varga,† Rita Szekely,† Monika Sebestyen,‡
́
́
́
́
́
́
́
Zsuzsa Jaszay,‡ Szabolcs Beni,§ Zoltan Nemes,§ Jean-Luc Pirat,|| Jean-Noel Volle,|| David Virieux,||
́
́
́
̈
⊥
#
∇
∇,○
Susan Szathmary,# Gyorgy Keri,†,◆
́
́
́
Agnes Gyuris, Katalin Kelemenics, Eva Ay, Janos Minarovits,
and Laszlo Orfi
†Vichem Chemie Ltd., Budapest 1022, Hungary
́
̈
†,§
̋
́
́
‡Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest 1111, Hungary
§Department of Pharmaceutical Chemistry, Semmelweis University, Budapest 1092, Hungary
||Institut Charles Gerhardt UMR5253, AM2N, ENSCM, Montpellier 34296 cedex 5, France
⊥RT Europe Research Center, Mosonmagyarov
#GalenBio Ltd., Mosonmagyarov
ar
́ ́
9200, Hungary
ar
́
́
9200, Hungary
∇Microbiological Research Group, National Center for Epidemiology, Budapest 1097, Hungary
○Faculty of Dentistry, Department of Oral Biology and Experimental Dental Research, University of Szeged, Szeged 6720, Hungary
◆MTA-SE Pathobichemistry Research Group, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry,
Semmelweis University, Budapest 1094, Hungary
S
* Supporting Information
ABSTRACT: Although there is a significant effort in the design of
a selective CDK9/CycT1 inhibitor, no compound has been proven
to be a specific inhibitor of this kinase so far. The aim of this
research was to develop novel and selective phosphorus containing
CDK9/CycT1 inhibitors. Molecules bearing phosphonamidate,
phosphonate, and phosphinate moieties were synthesized.
Prepared compounds were evaluated in an enzymatic CDK9/
CycT1 assay. The most potent molecules were tested in cell-based
toxicity and HIV proliferation assays. Selectivity of shortlisted
compounds against CDKs and other kinases was tested. The best compound was shown to be a highly specific, ATP-competitive
inhibitor of CDK9/CycT1 with antiviral activity.
highlighted below.8,9 The first indication, which is the most
INTRODUCTION
■
important disease for this study is HIV-1 infection.10 The second
The development of kinase inhibitors remains an area of
significant interest in drug discovery. To date, the FDA has
area with growing indication is cancer, where the use of selective
approved 29 small molecule drugs targeting kinases.1,2 Cyclin
CDK9 inhibitors may provide an appropriate strategy to drive
cancer cells into apoptosis.11−13 The third area is chronic
dependent kinases (CDKs) are serine/threonine kinases, which
activation of CDK9 which may cause cardiac hypertrophy and a
belong to a family of 13 protein kinases that share dependence
upon the binding of a cyclin subunit for activation.3 These
predisposition to heart failure, therefore the inhibition of CDK9
may constitute a new opportunity for a curative treatment.14
kinases are valid targets for drug development because their role
Finally, CDK9 also acts on several cytokines involved in the
was revealed in many proliferative disorders (e.g., cancer) and
inflammatory diseases.4 The cell cycle is controlled by the
regulation of inflammation, thus, CDK9 is considered as a
promising target for the treatment of rheumatoid arthritis and
activation and deactivation of CDKs. Consequently, the different
CDKs may be drug targets in a range of malignancies. To our
knowledge, only a few molecules have been reported as selective
inhibitors of CDKs and most of them inhibit at least two of the
CDKs by the same order of magnitude. Therefore, there remains
a need for more selective, or specific, inhibitors of the members of
the CDK family.5−7
chronic inflammatory diseases such as multiple sclerosis or
psoriasis.15
CDK9 and its cyclin partners (cyclin (Cyc) T1, T2a, b, and K)
form dimers upon binding. The CDK9/CycT1 is the most
common complex and plays a crucial role in HIV-1 replication.16
CDK9 is known to be involved in the molecular pathomechan-
isms of several diseases; the four most significant maladies are
Received: November 12, 2013
Published: April 17, 2014
© 2014 American Chemical Society
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Figure 1. Structure of some representative sulfonamide CDK9 inhibitors (1−5), the flavopiridol 6, and general structures of the presented phosphorus
moieties. References: 1 and 3,5 2 and 4,29 5,21 6.6
The most important role of CDK9/CycT1 is in the
phosphorylation of the C-terminal domain of RNA polymerase
II, which leads to the transcription of the viral genome.10 With
the exception of the viral Tat protein, the other associated
proteins are members of the transcription machinery of the host
cell. The virus hijacks the host cell signaling to turn it to its own
benefit. In this context, inhibition of CDK9 could halt or, at least,
slow down the HIV propagation.17
CDK9 shows high level of sequence conservation compared
with other members of the CDK family, thus the design of
selective CDK9 inhibitors is highly challenging.18 The first
CDK9 inhibitor involved in clinical trial was flavopiridol, and it
was discontinued due to its toxic effects.6 In our previous work,
we described several molecules showing remarkable CDK9
inhibition and high level of selectivity over other CDKs.5 At that
time, they were among the first selective CDK9 inhibitors.
Recently, other research groups have published highly potent
molecules, however, these compounds have significant effects on
other kinases as well.19,20 During the course of our work, Albert
et al. published a highly selective CDK9 inhibitor also based on
the 4-aminophenyl-6-phenylpyrimidine scaffold.21 These facts
maintained our interest to synthesize more selective and potent
CDK9 inhibitors containing a phosphorus subunit.
Resemblance of phosphorus containing moieties (e.g.,
phosphonamidates, phosphonates, and phosphinates) to sulfo-
namides and sulfonyl derivatives is obvious. Their similar shape
and electron density make these two types of functionalities to
ideal structural mimics of each other.22,23 Although phosphona-
midates, phosphonates, and phosphinates provide higher
substituent variability than sulfonyl moieties, they are rarely
applied in medicinal chemistry to replace sulfur containing grups.
Instead, sulfonamides and sulfonyl derivatives are widely used as
mimics of natural phosphate groups.24,25 Nonetheless, phospho-
nates and phosphinates were shown to be potent inhibitors of
peptidases as stable tetrahedral mimics of the transitional state
species of peptides,26 mimics of phosphorus containing
biomolecules involved in signal transduction27 and other
biomolecules.28
Using the isosteric relationship between sulfonamides and
phosphonamidates, phosphonates, and phosphinates, we antici-
pated the synthesis of phosphorus containing analogues of
previously published sulfonamide CDK9 inhibitors 1−5 (Figure
1). Our objective was to synthesize potent kinase inhibitors in
which a phosphorus moiety is essential to their inhibitory effect.
We aimed to improve selectivity toward the target kinase by
using these rarely applied functional groups and planned to
demonstrate in vitro efficacy on an HIV infected cell line.
CHEMISTRY
■
Inspired by the success of our sulfonamide-based CDK9
inhibitors and facing the incomplete selectivity toward the target
enzyme, we developed new synthetic approaches of phospho-
namidates, phosphonates, and phosphinates targeting CDK9.
They were prepared and evaluated against the corresponding
known sulfonamides 1−5 in biochemical assays. In the enzymatic
assays, flavopiridol 6 was used as a reference (Figure 1).
Synthesis of Phosphonamidate Inhibitors (Ar-NH-
P(O)ROR′). The aniline core was prepared according to
published methodologies, and subsequently the primary amino
group of 9 was functionalized with the appropriate phosphono-
chloridates 8a−c.5 The phosphonochloridates were prepared by
a two-step sequence involving the Arbuzov reaction of ethyl or
isopropyl bromide with triethyl or triisopropyl phosphite,
respectively, followed by the monochlorination of phosphonates
7a−c with oxalyl chloride (Scheme 1). The reaction of the
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Scheme 1. Synthesis of Phosphonic Acid Chlorides, Preparation of Phosphonamidates 10−12
a
Scheme 2. Synthesis of Phenol Derivatives, Phosphorylation, and Hydrolysis
a
Reagents and conditions: (a) IPA, cat. HCl, reflux; (b) t-BuOK, THF, RT; (c) SnCl2, EtOH, reflux; (d) Me3SiCl, KI, acetone, reflux.
primary amino group of 9 with phosphonochloridate 8a−c in
anhydrous pyridine led to the phosphonamidates 10−12 in 9−
18% yields. Because of the pH sensitivity of the P−N bond,
phosphonamidates were readily hydrolyzed upon workup.30
Synthesis of Phosphonate Inhibitors (Ar-O-P(O)ROR′).
Phenols 15−20 were readily accessible by the condensation of 4-
chloro-6-phenylpyrimidines 13a−b with aniline derivatives
14a−c (47−76%) in the presence of potassium tert-butoxide.
Phosphonates 21−29 were also synthesized by the reaction with
phosphonochloridates 8a−c (9−81%) in better yields compared
to phosphonamidates (Scheme 2). The nitro group of
phosphonates 22, 25, and 28 (Z = NO2) was consecutively
reduced to the corresponding amines 23, 26, and 29 by tin(II)
chloride in 21−81% yields. Finally, both phosphonic acid
analogues 30a and 30b were prepared by selective hydrolysis
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a
Scheme 3. Synthesis of meta- and para-Benzylphosphonate Intermediates
a
Reagents and conditions: (a) TEA, toluene, RT; (b) HCOOH·NH3, Pd/C, MeOH, RT; (c) MeSO2Cl, TEA, THF, RT; (d) NH2−NH2, Pd/C,
MeOH, RT; (e) cat. H2SO4, EtOH, reflux; (f) LiAlH4, THF, RT; (g) PBr3, CH2Cl2, RT; (h) P(OEt)3, neat, 140 °C; (i) HCOOH·NH3, Pd/C,
MeOH, RT.
a
Scheme 4. Synthesis of meta- and para-Benzylphosphonates and Phosphonic Acids
a
Reagents and conditions: (a) IPA, cat. HCl, reflux; (b) SnCl2, EtOH, reflux; (c) (1) HCl (5 M), reflux, (2) propylene oxide, EtOH, 0 °C.
of the corresponding alkyl phosphonates 21a and 21b using
TMSCl and KI in 70% and 79% yields, respectively.
The acid-catalyzed condensation of 4-chloro-6-phenylpyrimi-
dines 13a−b with anilines 33−37 was readily accomplished in
refluxing 2-propanol, furnishing benzylphosphonates 38, 39, 41,
42, and 44 in 6% to almost quantitative yields (Scheme 4).
Subsequently, the nitro derivatives 39a−b and 42a were reduced
with tin(II) chloride in refluxing ethanol, giving anilines 40a−b
and 43 (65−80%), respectively. Hydrolysis of phosphonate
esters 42a and 43 was achieved in refluxing 5 M hydrochloric
acid, affording 45a and 45b in 66% and 90% yield. The
hydrochloride salts were converted into the free bases by
treatment with propylene oxide (Scheme 4).
Synthesis of Phosphinates Inhibitors (Ar-CH2-P(O)-
ROR′). Precursor 46 was accessible in three steps from a
Michaelis−Becker reaction of 3-nitrobenzyl bromide with
phenyl-H-phosphinate (Scheme 5), followed by reduction of
the nitro group with tin(II) chloride; overall yield was 35%.
During the Michaelis−Becker reaction, the cleavage of the ethyl
ester was observed, necessitating an additional re-esterification
step with triethylorthoformate. Other precursors were prepared
Synthesis of Benzylphosphonate Inhibitors (Ar-CH2-
P(O)(OR)2). To obtain hydrolytically stable phosphonates where
the nitrogen or the oxygen atoms were replaced by a carbon,
benzyl derivatives were synthesized (Scheme 3). The first
reaction of the sequence was a Pudovik addition of diethyl
phosphite to the 3- or 4-nitro-benzaldehyde with diethyl
phosphite in 76% and 60% yields.31 On one hand, the Pudovik
adducts 31 and 32 were reduced to the corresponding amines 33
and 34 by transfer hydrogenation using ammonium formate and
Pd/C (y = 72 and 86%). On the other hand, mesylation of 31 and
32 (y = 73 and 52%), followed by reduction with hydrazine and
Pd/C, gave the benzylphosphonates 35 and 36 in 78% and 94%
yields (Scheme 3). Alternatively, 3-ethylphosphonomethyl-4-
methylaniline 37 was prepared from 2-methyl-5-nitrobenzoic
acid in five steps due to the limited availability of 2-methyl-5-
nitrobenzaldehyde (Scheme 3).
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a
Scheme 5. Synthesis of meta- and para-Benzylphosphinate Intermediates
a
Reagents and conditions: (a) neat, 140 °C; (b) HC(OEt)3, RT; (c) SnCl2, EtOH, reflux.
a
Scheme 6. Synthesis of meta- and para-Benzylphosphinates and Phosphinic Acids
a
Reagents and conditions: (a) IPA, cat. HCl, reflux; (b) SnCl2, EtOH, reflux; (c) Me3SiCl, KI, CHCl3, reflux.
by another pathway starting by the Arbuzov reaction between the
corresponding nitrobenzyl bromides and phosphonites,32
followed by the reduction of the nitro group with tin(II)
chloride, furnishing the desired aniline intermediates 47−51 in
28−70% overall yield.
Anilines 46−51 were reacted with 4-chloro-6-phenylpyrimi-
dines 13a−b under acidic conditions to give a series of
benzylphosphinates 52, 53, 55, 56, 58, and 59 in yields ranging
from 29 to 55%. After reduction of the nitro group with tin(II)
chloride, the corresponding anilines 54, 57, and 60 were
obtained in 77−88% yield. Phosphinic acid 61 was prepared by
cleavage of the ester function (52b) with trimethylsilyl chloride
in the presence of potassium iodide (Scheme 6).
14−64%).33 Nitro-phenylphosphonates and phosphinates were
reduced by tin(II) chloride to afford anilines in 49−90% yields
(Scheme 7).
Scheme 7. Synthesis of Anilinophosphinates and
a
Phosphonates
Synthesis of Arylphosphinate (Ar-P(O)ROR′) and
Arylphosphonate (Ar-P(O)(OR)2) Inhibitors. To further
explore the SAR of this compound family, it was also planned
to directly connect phosphonate and phosphinate moieties to the
aromatic ring without NH, O, or methylene spacers. The
phosphorus containing group is isosteric with the previously
presented scaffolds but closer in proximity to the aromatic ring.
Nonetheless, they can provide key information to the structure−
activity relationship.
Synthesis of arylphosphonate and phosphinate intermediates
was accomplished through a palladium catalyzed coupling
reaction between 3-iodo-nitro- or 4-bromo-nitrobenzene and
four different phosphinate or phosphonate substrates 62a−d (y =
a
Reagents and conditions: (a) Pd(PPh3)4, Et3N, toluene, reflux; (b)
SnCl2, EtOH, reflux.
Next, reactions of anilinophosphonates or phosphinates 63−
64 with 4-chloro-6-phenylpyrimidines 13a−b led to the N-
substituted analogues 65−74 (20−80% yields, Scheme 8).
Reduction of the nitro group by tin(II) chloride provided the
corresponding anilines 67, 70, and 75 in 59−97% yields.
Deprotection of phosphonic acids was accomplished by refluxing
in hydrochloric acid followed by neutralization using propylene
oxide (y = 22−69%).
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a
Scheme 8. Synthesis of meta- and para-Arylphosphinates and Arylphosphinates
a
Reagents and conditions: (a) IPA, cat. HCl, reflux; (b) SnCl2, EtOH, reflux; (c) (1) HCl (5 M), reflux, (2) propylene oxide, EtOH, 0 °C.
a
Scheme 9. Synthesis of 6-Phenyl Analogues
a
Reagents and conditions: (a) Pd(PPh3)4, Na2CO3, DME, H2O, RT, reflux; (b) IPA, cat. HCl, reflux.
a
Scheme 10. Synthesis of the Compounds with the Optimal Substituent Combination
a
Reagents and conditions: (a) IPA, cat. HCl, reflux; (b) Me3SiCl, KI, CHCl3, reflux.
Modifications on the 6-Phenyl Ring. Until now, no
variation was made on the phenyl ring apart from the methoxy,
nitro, and amino substituents. With this in mind, the effect of a
wider range of substituents on biological activity was
investigated. Targeted compounds 80−90 were prepared in
two steps from previously synthesized precursors (Scheme 9).
First of all, 4-chloro-6-phenylpyrimidines 13c−m were prepared
by the Suzuki coupling of the appropriate boronic acids and 4,6-
dichloropyrimidine in yields ranging from 20 to 80%.34
Synthesized intermediates 13c−m were then reacted with
anilinophosphinate 48, affording aminopyrimidines 80−90 in
10−67% yields. This aniline was preferred because its
preparation was more convenient than the ethyl counterpart 47.
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Benzyloxyphenyl-chloropyrimidine 13g was coupled with the
aniline 47 (y = 32%). Then the phosphonic esters 84 and 91 were
hydrolyzed to acids 92 and 93 in moderate yield (y = 32 and 19%,
respectively), Scheme 10.
Table 2. In Vitro Activity Phosphonates and Phosphonic
Acids
Preparation of phosphorus containing compounds is usually
more difficult than the corresponding sulfonamide analogues,
which led sometimes to low yields. Although, there were some
analogies between the different reactions applied in this work, the
yields were strongly affected by the different substitutions. The
conditions for the condensation reaction of 4-chloro-6-phenyl-
pyrimidines with anilines were developed as a general method.
However, conversions and yields of this reaction highly
depended on the nucleophilicity of the aniline, moreover,
minor modifications on the phosphorus groups showed a strong
influence on the reactivity of anilines.
CDK9/CycT1
IC50 (nM)
R1
R2
Me
R
R′
Et
Z
21a
21b
21c
22a
22b
23a
23b
24a
24b
24c
25a
25b
25c
26a
26b
26c
27a
27b
27c
28a
28b
29a
29b
30a
30b
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
H
Et
2-MeO
2-MeO
2-MeO
3-NO2
3-NO2
3-NH2
3-NH2
2-MeO
2-MeO
2-MeO
3-NO2
3-NO2
3-NO2
3-NH2
3-NH2
3-NH2
2-MeO
2-MeO
2-MeO
3-NO2
3-NO2
3-NH2
3-NH2
2-MeO
2-MeO
813
3057
Me
n-Pr
Ph
i-Pr
Et
Me
4137
Me
Et
Et
>10000
>10000
870
Me
n-Pr
Et
i-Pr
Et
RESULTS AND DISCUSSION
■
Me
For a comparison of phosphorus-based inhibitors with
sulfonamide derivatives, an in vitro test was conducted in first
intention. The kinase inhibitory profiles of the most potent
derivatives were determined, and then the efficiency of this small
set of compounds was validated on HIV infected cells. Finally,
kinetics of kinase inhibition was studied.
Me
n-Pr
Et
i-Pr
Et
5385
H
400
H
n-Pr
Ph
i-Pr
Et
1180
H
3100
H
Et
Et
>10000
>10000
>10000
1800
H
n-Pr
Ph
i-Pr
Et
In vitro enzymatic assays showed that phosphonamidates 10−
12 (Table 1) were significantly weaker inhibitors of CDK9 than
H
H
Et
Et
H
n-Pr
Ph
i-Pr
Et
6430
Table 1. In Vitro Activity of Phosphonamidates
H
5330
P-group
P-group
P-group
P-group
P-group
P-group
P-group
Me
Et
Et
360
H
n-Pr
Ph
i-Pr
Et
407
H
2840
H
Et
Et
>10000
>10000
3840
H
n-Pr
Et
i-Pr
Et
H
H
n-Pr
Et
i-Pr
H
6060
P-group
P-group
1490
R
R′
CDK9/CycT1 IC50 (nM)
Me
n-Pr
H
1457
10
11
12
Et
H
1450
3170
5240
n-Pr
Me
H
Ph
inactive, but a moderate activity was found with 42b and 45a
(Table 3). Substitution on the methylene spacer was not
beneficial. The phosphorus pattern attached to the meta-position
gave a better biological response than their para-counterparts.
Benzylphosphonate 44 with methyl substituent in the para-
position (IC50 = 5220 nM) was only a weak inhibitor of CDK9.
In this series, biological activity of phosphonic acid derivatives
45a and 45b increased in comparison to their ethyl ester
counterpart molecule 42a and 43. The most potent benzyl-
phosphonates 38 and 40a (IC50 = 333 and 332 nM, respectively)
were better than their phosphonate counterparts 24a and 26a
(IC50 = 400 and 1800 nM, Table 2), respectively.
Replacement of the phosphonate by a phosphinate group
resulted in a significant increase in potency (Table 4). The 3-
nitro-phenyl derivatives 53, 56, and 59 were found to be weak
inhibitors, in accordance with previous results reported in both
phosphonate series (Table 2−3). Once again, compounds of the
2-methoxy series possessed the best bioactivities among the 2-
methoxy, 3-nitro, and 3-amino derivatives. Ethyl and propyl
substitution on the phosphinate did not make a significant
difference, whereas the bigger phenyl group conferred lower
activity. meta-Phosphinates were shown to be more potent than
their para-equivalents. Hydrolysis of the ester 52b to 61 acid
slightly decreased the potency. For instance, compound 52a
exhibited an IC50 lower than 200 nM, whereas its phosphonate
the corresponding sulphonamides (compounds 1−5, Figure 1).
Their low stability toward hydrolysis prompted us to focus our
attention on the synthesis of phosphonate derivatives.
Potency of phosphonates 21a−30b showed to be deeply
dependent on the substituents of aromatic rings and on the
phosphorus atom (Table 2). Not surprisingly, all the nitro-
derivatives 22a−b, 25a−c, and 28a−b were not active at all. 2-
Methoxy-derivatives (21a−c, 24a−c, and 27a−b) were shown to
be more active than the corresponding 3-aminophenyl analogues
(23a−b, 26a−b, and 29a−b). The position of the phosphonate
group did not considerably influence the inhibitory effect,
meanwhile the size of phosphorus substituents on CDK9
inhibition followed the order: ethyl > propyl > phenyl. Free
phosphonic acids have a negative charge at physiologic pH; the
comparison between the alkyl ester 21a and the acid 30a showed
that the charged species is less active than the neutral one.
Comparison of ester 21b and acid 30b showed increasing
potency. Overall, three phosphonate inhibitors 24a, 27a, and 27b
achieved an IC50 close to 400 nM.
Methylene spacer between the phenyl ring and the
phosphonate group improved biological activity. Methoxy- (38,
41a−b) and aminophenyl (40a−b, 43) compounds were the
most potent ones. Nitrophenyl derivatives 39a−b and 42a were
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than 300 nM, and all of them contained the phosphorus
substituent in the meta-position.
Table 3. In Vitro Activity of Benzylphosphonates and
Benzylphosphonic Acids
When the phosphorus atom was directly bounded to the
aniline ring, no improved biological activity was observed both in
arylphosphinate and arylphosphonate series (Table 5). The 2-
Table 5. In Vitro Activity of Arylphosphinates and
Phosphonates
CDK9/CycT1
IC50 (nM)
R1
R2
R
R′
Z
38
P-group
P-group
P-group
P-group
P-group
H
H
H
H
H
H
H
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
H
2-MeO
3-NO2
3-NO2
3-NH2
3-NH2
2-MeO
2-MeO
3-NO2
3-NO2
3-NH2
2-MeO
3-NO2
3-NH2
333
9870
>10000
332
39a
39b
40a
40b
41a
41b
42a
42b
43
H
OH
H
OH
H
1401
490
CDK9/CycT1
IC50 (nM)
R1
R2
R
R′
Z
P-group
P-group
P-group
P-group
P-group
Me
H
OH
H
510
65
P-group
H
H
H
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
H
2-MeO
3-NO2
3-NH2
2-MeO
2-MeO
2-MeO
3-NO2
3-NO2
3-NO2
3-NH2
3-NH2
3-NH2
2-MeO
3-NO2
2-MeO
3-NO2
3-NH2
2-MeO
3-NO2
2-MeO
3-NH2
842
>10000
9802
H
>10000
4577
3110
5220
2100
1130
66
P-group
Et
H
OH
H
67
P-group
Et
H
68a
68b
68c
69a
69b
69c
70a
70b
70c
71
H
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
H
Et
418
44
P-group
H
H
H
n-Pr
Ph
1149
45a
45b
P-group
P-group
H
H
523
H
H
H
H
Et
>10000
>10000
>10000
>10000
>10000
6501
H
n-Pr
Ph
Table 4. In Vitro Activity of Benzylphosphinates and
Benzylphosphinic Acid
H
H
Et
H
n-Pr
Ph
H
P-group
P-group
H
EtO
EtO
EtO
EtO
EtO
OH
OH
OH
OH
1040
72
H
>10000
1020
73
P-group
P-group
P-group
H
74
H
>10000
>10000
429
75
H
76
P-group
P-group
H
CDK9/CycT1
IC50 (nM)
R1
R2
R
R′
Z
77
H
H
4100
78
P-group
P-group
H
580
52a
52b
52c
53a
53b
53c
54a
54b
54c
55a
55b
56a
56b
57a
57b
58
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
P-group
H
H
H
H
H
H
H
H
H
H
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
H
2-MeO
2-MeO
2-MeO
3-NO2
3-NO2
3-NO2
3-NH2
3-NH2
3-NH2
2-MeO
2-MeO
3-NO2
3-NO2
3-NH2
3-NH2
2-MeO
3-NO2
3-NH2
2-MeO
197
296
79
H
H
3320
n-Pr
Ph
517
Et
6432
8056
>10000
327
methoxy substituted derivatives were found to be the best
inhibitors regardless of the substituent R or the phosphorus
position. Compounds bearing a nitro group were once again
weak inhibitors, apart from phosphonic acid 77, which had a
moderate activity (IC50 = 4 100 nM). Surprisingly, none of the 3-
amino analogues exhibited a considerable inhibitory effect on
CDK9 activity. In this series, phosphonic acids (76−79) had
significantly lower IC50s than their ester counterparts (71−73,
75). Again, the ethylphosphinate derivative (68a, IC50 = 418
nM) was shown to be the most potent inhibitor of this series.
Substituent variations on the 6-phenyl ring provided a clear
structure−activity relationship. In general, substitutions in
position 2 were preferred (Table 6). Kinase inhibition was
optimum with inhibitors containing 2-alkoxy substituents and
weakly dependent on the nature of the alkyl substituent.
Meanwhile, any substituents in other positions caused a
significant decrease of biological activity. If these alkoxy groups
were replaced by an alkyl substituent (90) or were moved to
another position (85, 86, 89), the activity significantly decreased.
This clearly showed that an alkoxy group is essential in the
position 2.
n-Pr
Ph
Et
n-Pr
Ph
682
1019
743
P-group
P-group
P-group
P-group
P-group
P-group
Me
Et
H
n-Pr
Et
652
H
7774
5525
7533
7474
366
H
n-Pr
Et
H
H
n-Pr
Et
P-group
P-group
P-group
P-group
59
Me
Et
7022
346
60
Me
Et
61
H
n-Pr
261
counterpart 24a was 2-fold higher (400 nM). Similarly, the
methyl substituted analogue 58 (IC50 = 366 nM) was 4-fold more
potent than the phosphonamidate 10 (IC50 = 1 450 nM) and 2-
fold more potent than the phosphonate 21a (IC50 = 813 nM).
Finally, three molecules 52a, 52b, and 61, showed IC50 lower
On the basis of the results obtained from this structure−
activity study, a fragment-based approach was applied involving
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The 3-amino moiety was slightly weaker in activity than the 2-
alkoxy, while the 3-nitro and 2-alkyl moieties were inactive.
Among the 2-alkoxy moieties, the trend was the bigger the
substituent, the lower the IC50.
Table 6. In Vitro Activity of Phosphinates with 6-Phenyl
Variations
One of the aims of this project was to construct a potent kinase
inhibitor whose phosphorus moiety was crucial to its inhibitory
effect, and this goal was achieved. The examination of the
chemical stability of the phosphorus moiety provided the
evidence that in the case of the phosphonamidates, it is probable
that the P−N bond was cleaved at non-neutral pH. In this case, a
low IC50 of aniline would have been expected,5 however, it was
not the case. In the case of phosphonates, hydrolysis of the P−O
bond may occur, however, good kinase inhibitory effects of
phenols were not observed.5 In the case of benzyl and
arylphosphinates and phosphonates, the possibility of hydrolysis
may be excluded.
Selectivity of Inhibitors. We aimed to synthesize selective
CDK9 inhibitors. For this purpose, inhibitory activity of the best
compounds was measured in an in vitro activity assay against the
CDK2/CycE and the CDK4/CycD1, which are two of the most
important regulators of the cell cycle among the CDKs. CDK9
selectivity was improved from phosphonates to phosphinates
(Table 8), and two compounds (52a, 52b) showed almost
complete CDK9 inhibition with moderate effects on CDK2 and
CDK4.
Z
CDK9/CycT1 IC50 (nM)
80
81
82
83
84
85
86
87
88
89
90
2-EtO
254
248
2-n-PrO
2-i-PrO
2-PhO
210
601
2-BnO
143
3-MeO
4-MeO
2,3-diMeO
2-MeO-4-F
3-BnO
6937
5414
2387
186
>10000
>10000
2-Et
identified groups which gave high inhibitory activity.35 On the 4-
aminophenyl ring, the optimal substituent was the ethyl-
phosphinate derivative in the meta-position (compound 52a,
IC50 = 197 nM). On the terminal 6-phenyl ring, the best
biological result was observed with an ortho-benzyloxy
substituent (compound 84, IC50 = 143 nM). Combination of
these functionalities (compound 91) had a slightly weaker
activity (IC50 = 212 nM) compared to its methoxyphenyl
counterpart 52a. Moreover, it was less active than the
propylphosphinate derivative 84. After hydrolysis of ester 91,
its activity increased considerably (compound 92; IC50 = 163
nM). Interestingly, no change in activity was observed after the
hydrolysis of ester 84 to the phosphinic acid 93 (IC50 = 143 and
142 nM, respectively; Table 7).
a
Table 8. CDK Selectivity Profile of Selected Compounds
CDK2/CycE
inhibition (%)
CDK4/CycD1
inhibition (%)
CDK9/CycT1
inhibition (%)
6
99
32
19
1
84
2
100
83
11
12
18
13
21
19
46
51
68
30
24*
14*
22*
22
35
40
67
71
21a
21b
21c
30b
41a
41b
43
100
83
6
8
75
34
77
94
14
36*
23*
18*
69
18
56
98
88
98
Table 7. In Vitro Activity of the Phosphinates with the Best
Substituent Combinations
97
80
52a
52b
54a
73
98*
95*
96*
106
43
75
76
91
79
100
a
R
R′
CDK9/CycT1 IC50 (nM)
Compound concentration was 10 μM, except * 1 μM.
91
84
92
93
Et
Et
Et
H
H
212
143
163
142
n-Pr
Et
As a consequence, lead compounds 52a and 52b were further
investigated on a panel of 30 kinases in in vitro activity assays.
Results of this screen suggested outstanding selectivity: only 52a
shows moderate inhibition on cKit and Ret kinases, 28 and 34%
inhibition at 1 μM, respectively (Table 9).
The most potent CDK9 inhibitor, compound 93, was
subjected to a wider selectivity profiling against 451 kinases in
binding assays. A similar kinase profiling experiment has already
been published with flavopiridol 6 on 442 kinases.36 We used
these results as a basis for comparison. Compound 93 showed
noticeable affinity toward only IRAK3 (18% of the control at 1
μM concentration), which means that its selectivity score is S(%
Ctrl <35) = 0.003 for nonmutant kinases (Figure 2).
Interestingly, this compound does not bind to the inactive
n-Pr
The development of a new drug candidate through the
fragment-based approach is an iterative process, and some
general structure−activity relationships stand out from these
results. First, the ethyl substituted phosphonamidate, phospho-
nates, and phosphinates were the best inhibitors. Second, the
smaller the substituent on the phosphorus atom, the higher the
biological effect observed. Phosphinates with methylene spacer
in the meta position were shown to be the most potent inhibitors.
Examination of substituent variations on the 6-phenyl ring
indicated that 2-alkoxy substitutions were the most beneficial.
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a
Table 9. Kinase Selectivity Profile of Selected Compounds
ABL
AKT1
AXL
bRAF
CDK2/CycE
CDK4/CycD1
CDK9/CycT1
CHK1
cKIT
cMET
CSK
52a
52b
20
9
11
21
7
0
5
36
23
24
98
95
12
14
28
16
22
6
24
14
−1
14
DDR1
ERBB2
FGFR3
FLT3
IKKβ
INSR
IRAK4
JAK3
JNK1
mTOR
PAK1
52a
52b
13
7
11
5
8
12
6
15
7
12
2
16
9
9
0
19
8
3
9
20
10
1
PAK4
PDGFRb
PIM1
PKCa
RET
ROCK2
SRC
SYK
TIE2
TrkA
VEGFR2
52a
52b
25
19
18
2
8
8
9
34
16
14
7
18
4
8
10
5
8
11
6
−2
−1
−1
a
Inhibition (%) at 1 μM compound concentration.
Figure 2. Kinase selectivity profiles of flavopiridol (Kd ≤ 1 μM; CDK9 is highlighted with blue)38 and compound 93.
form of CDK9,37 which was used in this assay. It does, however,
Table 10. Cell Viability on MT4 Cells after 7 Days Incubation
inhibit the activity of CDK9/CycT1 in the nanomolar range.
According to these results the phosphonic acid 93 is a highly
specific inhibitor of the CDK9/CycT1 complex. Generally,
known CDK9 inhibitors inhibit other CDKs in the nanomolar
range. The most selective CDK9 inhibitor published so far is
LDC000067, which does not inhibit other CDKs, however, its
off-targets are GSK3A and HGK/MAP4K4.21 Although the
structural similarities between our compounds and LDC000067
are obvious (compound 5, Figure 1), specificity of compound 93
toward CDK9/CycT1 is higher.
Cell-Based Assays. Cytotoxic effect of selected compounds
was tested in vitro on MT4 human T lymphocyte cell line by
MTT cell proliferation assay. MT4 cells are ideal model for HIV-
1 infection experiments.39 After 7 days incubation with different
compound concentrations (2500 and 1250 nM), we identified
the proliferative activity of remaining cells. Viability was
determined as relative value to the untreated cells (100%).
Most of the compounds did not affect cell viability at 1250 nM;
moreover compound 93 seemed to be triggering cell
proliferation. The tested phosphonamidate 10, phosphonate
23a, and phosphonic acid 30a showed some toxic effect at 2500
nM concentration (Table 10). According to these results, 1250
nM concentration was chosen for the HIV-1 proliferation assay.
Given the fact that CDK9/CycT1 is a validated target in HIV-1
infection, the most potent compounds were tested in an HIV-1
proliferation assay. This experiment is a cell proliferation assay
viability (% of the ctrl)
2500 nM
1250 nM
10
29 26
108 62
76 22
74 14
100 20
94 24
96 22
78 16
72 20
78 14
91 11
94 18
21a
23a
30a
38
52a
52b
76
81
9
66
90
9
7
90 15
93 13
94 27
104 18
141 11
84
86 23
98 30
91
92
110 20
158 11
93
(MTT) on HIV-1 infected MT4 cells. Proliferation of virus
infected cells decreased compared to noninfected ones. If the
tested compound inhibited virus proliferation, higher prolifer-
ative activity would be observed compared to the untreated,
infected control.40 The HIV-1 infected untreated cells were
considered to be 100%. The best CDK9 inhibitors from each
group were tested. The treatment concentration was 1250 nM;
AZT (azidothymidine) was used as the positive control41 at 25
nM. Phosphonamidate 10, phosphonates 21a, 23a, and 40a, and
phosphonic acid 30a did not show any inhibition of HIV-1
proliferation. However, phosphinates (52a, 52b, 84, 91−93) did
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protect MT4 cells against viral infection similarly to the positive
control AZT. Higher viability correlated with lower CDK9 IC50.
Compound 76 did not fit to this trend. It was hypothesized that
this molecule, as a phosphonic acid, may bind and inhibit some
proteins other than CDK9 which are beneficial for HIV-1
proliferation inhibition (Table 11). This hypothesis is supported
by the lower selectivity of phosphonic and phosphinic acids (30b,
76, 79 in Table 8).
CONCLUSION
■
Different phosphinates, phosphonates, and phosphonamides
were prepared in a few steps from readily available starting
materials through affordable and general methods. They were
considered as bioisosteres of sulfonamide-based CDK9 inhib-
itors. In these series, the most potent inhibitor was the
phosphinic acid 93, which was proved to be highly specific to
CDK9/CycT1 and acts in an ATP-competitive manner.
Although its inhibitory activity is slightly lower than the best
CDK9 inhibitors in the literature, its high specificity for CKD9/
CycT1 is exceptional. These results suggested that 93 would have
the potential to control HIV-1 replication in vivo while having a
lower risk of inhibiting other kinases and consequently causing
undesired toxicity.
Table 11. HIV-1 Proliferation Assay in MT4 Cells
CDK9/CycT1 IC50
viability (rel % to infected, untreated cells)
(nM)
10
102 18
95 15
1453
813
21a
23a
30a
AZT
40a
52a
52b
76
In general, these results demonstrate the bioisosteric utility of
the phosphonamidate, phosphonate, and phosphinate moieties
in the design of novel kinase inhibitors.
99 23
870
96 20
1490
158 24
96 36
332
197
296
429
105 23
131 21
130 31
132 29
148 12
135 16
124 21
141 25
135 22
EXPERIMENTAL SECTION
■
Reagents and solvents were purchased from Sigma-Aldrich Co.,
AlfaAesar Ltd., or Apollo Ltd. and were used without further purification
or drying. Melting points were measured on a Buchi B-540 apparatus,
and measured data are uncorrected. For column chromatography,
Merck silica gel 60 (35−70 μm) was used or Silica gel 60 F254 1 mm
preparative thin layer plates were applied from Merck GmbH. NMR
spectra were recorded on different apparatus as it is indicated in the
assignation:
AZT
84
143
212
163
142
91
92
93
AZT
A Bruker AC 300 for H-300 MHz, 31P-121 MHz, 13C-75 MHz
1
measurements.
1
A Bruker AC 400 for H-400 MHz, 31P-162 MHz, 13C-101 MHz
measurements.
1
A Bruker AC 250 for H-250 MHz, 31P-101 MHz, 13C-63 MHz
Kinetics. For further development, it was beneficial to
determine the mechanism of kinase inhibition. We hypothesized
that these compounds were ATP-competitive inhibitors. To
prove this theory, the kinase inhibition was measured at different
ATP concentrations.42 The inhibitor concentration was kept at a
constant 260 nM in each experiment. Five ATP concentrations
were applied: the KmATP (for this enzyme it was 12 mM), two
lower (4 and 1.33 mM) and two higher (36 and 108 mM)
concentrations. If these compounds were ATP-competitive
inhibitors, the value of the Ki would be half of the measured
IC50 according to the Cheng−Prusoff equation,43 and the ratio of
IC50/Ki would be increased at higher ATP concentration.
According to the results (Figure 3), our compounds were shown
to be ATP-competitive inhibitors.
measurements.
A Varian Unity Inova 600 for 1H-600 MHz, 31P-242 MHz
measurements.
Chemical shifts are given in parts per million (δ) referenced to TMS
(δ = 0.00 ppm 1H-, 13C NMR) or 85% H3PO4 (δ = 0.00 ppm 31P NMR).
Coupling constants are given in hertz. Electrospray ionization mass
spectra were recorded on Waters 2795 HPLC, equipped with Waters
996 photodiode array detector and Micromass ZMD 2000 LC-MS
system, which were also used to determine the purity of certain
materials. Retention times concern to a Supelco Discovery C18, 5 cm ×
4.6 mm, 5 μm column. Eluents were water/0.05% HCOOH and
MeCN/0.05% HCOOH in gradient. Flow rate was 2 mL/min. Injected
quantity of each sample was 3 μg. MW-reactor: for 0,2−20 mL, a
Personal Chemistry, Emrys Creator; for 30−1000 mL, a Milestone Inc.,
Ethos MicroSYNTH Labstation reactor was used. Purity of prepared
compounds was determined by LC-MS, 1H and 31P NMR. All
compounds tested in biological assay were >95%. Purity of
intermediates was >90%, unless otherwise stated.
Chemistry. Ethyl Ethylphosphonochloridate (8a). Ethyl dieth-
ylphosphonate (0.84 mL, 5 mmol) and one drop of dry DMF were
dissolved in dry CH2Cl2 (5 mL). The mixture was cooled to 0 °C, and
oxalyl chloride (1.27 mL, 15 mmol) was added slowly. The mixture was
stirred at ambient temperature for 5−6 h then the volatiles were
evaporated and additional CH2Cl2 (10 mL) was evaporated two more
times to obtain 0.704 g (90%) as a brown oil. Product was used without
1
further purification. 31P NMR (CDCl3, 162 MHz): δ 46.7 ppm. H
NMR (CDCl3, 400 MHz): δ 4.10−4.30 (m, 2H), 2.00−2.15 (m, 2H),
1.28−1.38 (m, 3H), 1.13−1.27 (m, 3H).
Isopropyl Propylphosphonochloridate (8b). Triisopropyl-phos-
phite (12.3 mL, 50 mmol) and 1-bromopropane (18.2 mL, 200
mmol) were reacted in Arbuzov reaction at 150 °C for 4 h in a
microwave reactor. The product (7b) was purified by vacuum
distillation, and a colorless oil was obtained (6.551 g, 63%); bp = 72−
76 °C (3.35 mbar). 31P NMR (CDCl3,162 MHz): δ 30.3 ppm. 1H NMR
(CDCl3, 250 MHz): δ 4.60−4.70 (m, 2H), 1.55−1.70 (m, 4H), 1.20−
Figure 3. Determination of ATP competition of six inhibitors at 260 nM
inhibitor concentration.
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1.30 (m, 12H), 0.92−1.00 (m, 3H). The acid chloride was prepared with
the method described for the preparation of 8a; 0.830 g (90%) of a
brown oil was obtained. 31P NMR (CDCl3, 162 MHz): δ 43.2 ppm. 1H
NMR (CDCl3, 250 MHz): δ 4.80−4.95 (m, 1H), 1.95−2.13 (m, 2H),
1.55−1.80 (m, 2H), 1.20−1.40 (m, 6H), 0.90−1.05 (m, 3H).
Ethyl Phenylphosphonochloridoate (8c). Compound 8c was
prepared according to the method used for 8a, but the mixture was
refluxed for 24 h, giving a brown oil (0.920 g, 90%). Product 8c was used
without further purification. 31P NMR (CDCl3, 162 MHz): δ 29.3 ppm.
1H NMR (CDCl3, 250 MHz): δ 7.75−7.95 (m, 2H), 7.50−7.6 (m, 1H),
The combined organic layers were washed with 1 M aqueous NaOH (2
× 10 mL), with brine (30 mL), and dried with Na2SO4. After
concentration under vacuum, the crude product was further purified by
preparative TLC with 100% EtOAc as eluent. For the required purity,
two−three elutions were necessary. The desired product was washed
with MeOH from silica. If the product contained some traces of
phosphonic acid (coming from its hydrolysis on silica), it was dissolved
in CH2Cl2 (20 mL) washed with 1 M aqueous NaOH (20 mL), dried
with Na2SO4, and evaporated. The product was recrystallized from
MeCN.
7.35−7.5 (m, 2H), 4.25−4.45 (m, 2H), 1.43 (t, 3JHH = 7.1 Hz, 3H).
N1-[6-(2-Methoxyphenyl)pyrimidin-4-yl]-4-methylbenzene-1,3-di-
amine (9). 2-Methyl-5-nito aniline (1.52 g, 10 mmol) was dissolved in
dry pyridine (50 mL). Isobutyl chloroformate (1.42 mL, 11 mmol) was
added in one portion. The reaction mixture was stirred at RT overnight
then evaporated to dryness. The crude product was suspended in 1 M
HCl and the precipitate was filtered off and the resulting solid was dried
in a desiccator over P2O5, giving a yellow powder (1.915 g, 76%). If
necessary, it can be recrystallized from 2-propanol; mp = 118 °C (IPA).
1H NMR (CDCl3, 400 MHz): δ 8.74 (s, 1H), 7.8 (dd, 3JHH = 8.3 Hz,
Ethyl P-Ethyl-N-(5-{[6-(2-methoxyphenyl)pyrimidin-4-yl]amino}-
2-methylphenyl)phosphonamidate (10). Prepared by the reaction of
aniline 9 and phosphonochloridate 8a. Cream-colored powder (0.014 g,
17%); mp = ∼RT, amorphous. 31P NMR (DMSO-d6, 162 MHz): δ 32.1
ppm. 1H NMR (DMSO-d6, 400 MHz): δ 9.58 (s, 1H), 8.62 (d, 4JHH
1.1 Hz, 1H), 7.91 (dd, 3JHH = 7.7 Hz, 4JHH = 1.8 Hz, 1H), 7.56 (d, 4JHH
=
=
2.0 Hz, 1H), 7.42−7.48 (m, 1H), 7.41 (d, 4JHH = 1.1 Hz, 1H), 7.31 (dd,
3JHH = 8.3 Hz, 4JHH = 2.0 Hz, 1H), 7.17 (d, 3JHH = 7.7 Hz, 1H), 7.04−
7.10 (m, 2H), 6.48 (d, 2JPH PH
= 7.3 Hz, 1H), 3.95−4.01 (m, 2H), 3.88
J
(s, 3H), 2.19 (s, 3H), 1.75−1.90 (m, 2H), 1.25 (t, 3JHH = 6.8 Hz, 3H),
1.05 (dt, 3JPH = 19.7 Hz, 3JHH = 7.6 Hz, 3H). MS[ES+, Q-TOF]: M + H+
= 427.23 m/z; 2M + H+ = 853.35 m/z.
4JHH = 2.3 Hz, 1H), 7.23 (d, 3JHH = 8.6 Hz, 1H), 6.45 (s, 1H), 3.94 (d,
3JHH = 6.8 Hz, 2H), 2.29 (s, 3H), 1.95 (m, 1H), 0.92 (d, 3JHH = 6.8 Hz,
6H). The (2-methyl-5-nitro-phenyl)-carbamic acid isobutyl ester was
dissolved in MeOH (5 mmol in 100 mL), and 10% Pd/C (10% w/w)
was added to the solution. The mixture was stirred under atmospheric
pressure of H2. When the consumption of H2 was finished, the catalyst
was filtered off through a pad of celite and washed with MeOH. The
filtrate was evaporated to give (5-amino-2-methyl-phenyl)-carbamic
acid isobutyl ester as a pink or brown powder (∼80%); mp = 100−101
°C (MeOH). 1H NMR (CDCl3, 250 MHz): δ 7.34 (s, 1H), 6.94 (d, 3JHH
= 8.0 Hz, 1H), 6.38 (dd, 3JHH = 8.0 Hz, 4JHH = 2.3 Hz, 2H), 3.97 (d, 3JHH
Isopropyl N-(5-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-2-
methylphenyl)-P-propylphosphonamidate (11). Prepared by the
reaction of aniline 9 and phosphonochloridate 8b. Creamy colored
powder (0.008 g, 9%); mp = 151 °C (MeCN). 31P NMR (DMSO-d6,
1
162 MHz): δ 29.3 ppm. H NMR (DMSO-d6, 400 MHz): δ 9.52 (s,
1H), 8.62 (s, 1H), 7.92 (d, 3JHH = 6.1 Hz, 1H), 7.6 (s, 1H), 7.42−7.48
(m, 1H), 7.41 (s, 1H), 7.27 (d, 3JHH = 6.1 Hz, 1H), 7.17 (d, 3JHH = 8.6
Hz, 1H), 7.04−7.10 (m, 2H), 6.38 (d, 3JHH = 7.1 Hz; 1H), 4.57−4.67
(m, 1H), 3.89 (s, 3H), 2.19 (s, 3H), 1.70−1.90 (m, 2H), 1.48−1.60 (m,
2H), 1.24 (dd, 4JPH = 25.2 Hz, 3JHH = 6.1 Hz, 6H), 1.0 (dt, 4JPH = 40.6
Hz, 3JHH = 7.2 Hz, 3H). MS[ES+, Q-TOF]: M + H+ = 455.27 m/z.
Ethyl N-(5-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-2-meth-
ylphenyl)-P-phenylphosphonamidate (12). Prepared by the reaction
of aniline 9 and phosphonochloridate 8c. Creamy colored powder
(0.017 g, 18%); mp = 172 °C (MeCN). 31P NMR (DMSO-d6, 162
MHz): δ 17.2 ppm. 1H NMR (DMSO-d6, 400 MHz): δ 9.46 (s, 1H),
= 6.8 Hz, 2H), 3.05 (br, 2H), 2.17 (s, 3H), 2.00 (m, 1H), 0.99 (d, 3JHH
=
6.8 Hz, 6H). (5-Amino-2-methyl-phenyl)-carbamic acid isobutyl ester
(0.222 g, 1 mmol) and 4-chloro-6-(2-methoxy-phenyl)-pyrimidine 13a
(0.22 g, 1 mmol) were dissolved in 2-propanol (10 mL) then a solution
of HCl saturated 2-propanol (2 mL) was added to the mixture. The
reaction mixture was refluxed for 3−4 h then it was concentrated under
vacuum. The residue was suspended in satd NaHCO3 and extracted with
EtOAc:THF (2:1, 3 × 50 mL). The combined organics were washed
with brine and dried with Na2SO4. After evaporation of solvents under
reduced pressure, the crude product was recrystallized from MeCN to
give the {5-[6-(2-methoxy-phenyl)-pyrimidin-4-ylamino]-2-methyl-
phenyl}-carbamic acid isobutyl ester as a pale-yellow powder (0.304 g,
75%); mp = 155 °C (MeCN). 1H NMR (DMSO-d6, 400 MHz): δ 9.58
(s, 1H), 8.84 (s, 1H), 8.66 (d, 4JHH = 1.2 Hz, 1H), 7.95 (dd, 3JHH = 7.8
Hz, 4JHH = 1.8 Hz, 1H), 7.68 (d, 4JHH = 2.3 Hz, 1H), 7.47 (m, 2H), 7.43
(d, 4JHH = 1.2 Hz, 1H), 7.18 (d, 3JHH = 7.6 Hz, 1H), 7.14 (d, 3JHH = 8.8
Hz, 1H), 7.07 (td, 3JHH = 7.6 Hz, 4JHH = 1.0 Hz, 1H), 3.9 (s, 3H), 3.86 (d,
3JHH = 6.8 Hz, 2H), 2.17 (s, 3H), 1.92 (m, 1H), 0.94 (d, 3JHH = 6.8 Hz,
6H). MS[ES+, Q-TOF]: M + H+ = 407.25 m/z, 2M + H+ = 813.41 m/z.
The {5-[6-(2-methoxy-phenyl)-pyrimidin-4-ylamino]-2-methyl-phe-
nyl}-carbamic acid isobutyl ester and 5 mol equiv of KOH were
refluxed overnight in EtOH:water (4:1, 1 mmol in 50 mL). The reaction
mixture was concentrated under vacuum, and the residue was suspended
in water and 0.5 mL of EtOAc. This mixture was stirred for 15 min, and
then it was filtered off. The white powder was washed with Et2O (80%).
If necessary, it can be recrystallized from MeCN; mp = 168 °C (MeCN).
1H NMR (DMSO-d6, 400 MHz): δ 9.19 (s, 1H), 8.54 (s, 1H), 7.85 (dd,
3JHH = 7.6 Hz, 4JHH = 1.8 Hz, 1H), 7.37 (dd, 3JHH = 8.4 Hz, 4JHH = 1,8 Hz,
1H), 7.31 (d, 4JHH = 1.3 Hz, 1H), 7.08 (d, 3JHH = 7.6 Hz, 1H), 6.99 (dd,
3JHH = 7.6 Hz, 1H), 6.86 (s, 1H), 6.79 (d, 3JHH = 8.1 Hz, 1H), 6.66 (d,
3JHH = 7.8 Hz, 1H), 4.79 (br, 2H), 3.81 (s, 3H), 1.95 (s, 3H). MS[ES+,
Q-TOF]: M + H+ = 307,18 m/z.
8.58 (s, 1H), 7.91 (d, 3JHH = 9.2 Hz, 1H), 7.82 (dd, 3JPH = 12.8 Hz, 3JHH
=
7.2 Hz, 2H), 7.53−7.59 (m, 2H), 7.46−7.53 (m, 2H), 7.45 (t, 3JHH = 8.4
Hz, 1H), 7.36 (s, 1H), 7.26 (d, 3JHH = 7.7 Hz, 1H), 7.17 (d, 3JHH = 8.5
Hz, 1H), 7.08 (t, 3JHH = 7.4 Hz, 1H), 6.99−7.05 (m, 2H), 4.10 (dq, 3JPH
≈ 3JHH ≈ 7.2 Hz, 2H), 3.88 (s, 3H), 2.19 (s, 3H), 1.29 (t, 3JHH = 7.2 Hz,
3H). MS[ES+, Q-TOF]: M + H+ = 475.21 m/z; 2 M + H+ = 949.39 m/z.
4-Chloro-6-(2-methoxyphenyl)pyrimidine (13a) and 4-Chloro-6-
(3-nitrophenyl)pyrimidine (13b). These compounds were prepared
according to ref 5.
5-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-2-methylphenol
(15). 5-Amino-2-methyl-phenol 14a (0.682 g, 5.5 mmol) and 4-chloro-
6-(2-methoxy-phenyl)-pyrimidine 13a (1.10 g, 5.0 mmol) were
dissolved in 2-propanol (25 mL) then HCl saturated 2-propanol (5
mL) was added to the mixture. The reaction mixture was refluxed for 1 h
then it was evaporated. The residue was suspended in satd NaHCO3 and
extracted with EtOAc (3 × 50 mL). The combined organics were
washed with brine and dried with Na2SO4. After evaporation under
reduced pressure, the crude product was recrystallized from MeCN to
give a pale-yellow, cream-colored powder (0.924 g, 60%); mp = 220−
223 °C (MeCN). 1H NMR (DMSO-d6, 400 MHz): δ 9.42 (s, 1H, exch
with D2O), 9.32 (s, 1H, exch with D2O), 8.65 (d, 4JHH = 1.2 Hz, 1H),
7.93 (dd, 3JHH = 7.7 Hz, 4JHH = 1.8 Hz, 1H), 7.45 (td, 3JHH = 7.5 Hz, 4JHH
= 1.8 Hz, 1H), 7.40 (d, 4JHH = 1.1 Hz, 1H), 7.27 (s, 1H), 7.17 (d, 3JHH
=
7.8 Hz, 1H), 7.08 (td, 3JHH = 7.8 Hz, 4JHH = 1.0 Hz, 1H), 6.98 (m, 2H),
3.89 (s, 3H), 2.08 (s, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 160.4
(1C), 159.2 (1C), 157.8 (1C), 157.5 (1C), 155.3 (1C), 138.4 (1C),
131.0 (1C), 130.3 (1C), 130.1 (1C), 125.9 (1C), 120.5 (1C), 118.0
(1C), 112.0 (2C), 110.6 (1C), 106.7 (1C), 55.7 (1C), 15.5 (1C).
MS[ES+, Q-TOF]: M + H+ = 308.13 m/z.
General Procedure for the Preparation of Phosphonamidates. The
aniline 9 (0.061 g, 0.2 mmol) was dissolved in dry pyridine (5 mL) and
the appropriate phosphonic acid 8a−c (0.4 mmol) was added and the
mixture was stirred at 50 °C for 24 h under N2 atmosphere. The volatiles
were evaporated then the residue was suspended in satd NH4Cl aqueous
solution (10 mL). The product was extracted with CH2Cl2 (3 × 20 mL).
2-Methyl-5-{[6-(3-nitrophenyl)pyrimidin-4-yl]amino}phenol (16).
16 was prepared according to the procedure used for compound 15
starting from 5-amino-2-methyl-phenol 14a (0.677 g, 5.5 mmol) and 4-
3950
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Article
chloro-6-(3-nitrophenyl)-pyrimidine 13b (1.178 g, 5.0 mmol). 16 was
recrystallized from MeCN to give a yellow powder (0.757 g, 47%); mp =
240−245 °C (MeCN). 1H NMR (DMSO-d6, 300 MHz): δ 9.58 (s, 1H),
9.34 (s, 1H), 8.81 (s, 1H), 8.73 (s, 1H), 8.43 (s, 1H), 8.36 (s, 1H), 7.83
(s, 1H), 7.34 (s, 1H), 7.26 (s, 1H), 7.00 (s, 2H), 2.09 (s, 3H). Each peak
is a broad singlet. LC-MS: Rt = 3.44 min. (ESI): m/z = 323 [M + H]+.
3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenol (17). 17
was prepared according to the procedure used for compound 15
starting from 3-amino-phenol 14b (0.600 g, 5.5 mmol) and 4-chloro-6-
(2-methoxyphenyl)-pyrimidine 13a (1.100 g, 5.0 mmol). 17 was
recrystallized from MeCN to give a yellow powder (0.923 g, 63%); mp =
217 °C (MeCN). 1H NMR (DMSO-d6, 300 MHz): δ 9.5 (s, 1H), 9.38
(s, 1H), 8.68 (s, 1H), 7.95 (dd, 3JHH = 7.7 Hz, 4JHH = 1.7 Hz, 1H), 7.40−
7.50 (m, 1H), 7.46 (s, 1H), 7.28 (s, 1H), 7.17 (d, 3JHH = 8.2 Hz, 1H),
7.05−7.15 (m, 3H), 6.43 (d, 3JHH = 7.4 Hz, 1H), 3.90 (s, 3H). LC-MS:
Rt = 0.47 min; 1.98 min; 2.25 min. (ESI): m/z = 294 [M + H]+.
3-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenol (18). 18 was
prepared according to the procedure used for compound 15 starting
from 3-amino-phenol 14b (0.600 g, 5.5 mmol) and 4-chloro-6-(3-
nitrophenyl)-pyrimidine 13b (1.178 g, 5.0 mmol). 18 was recrystallized
from MeCN to give a yellow powder (1.17 g, 76%); mp = 250−252 °C
(MeCN). 1H NMR (DMSO-d6, 300 MHz): δ 10.31 (br, 1H), 9.5 (br,
1H), 8.82 (s, 1H), 8.78 (s, 1H), 8.43 (d, 3JHH = 7.8 Hz, 1H), 8.38 (d, 3JHH
= 7.8 Hz, 1H), 7.86 (t, 3JHH = 7.9 Hz, 1H), 7.58 (s, 1H), 7.33 (s, 1H),
7.08−7.33 (m, 2H), 6.5 (d, 3JHH = 7.8 Hz, 1H). LC-MS: Rt = 3.20 min.
(ESI): m/z = 309 [M + H]+.
(d, 3JPC = 2.9 Hz, 1C), 107.2 (1C), 62.1 (d, 2JPC PC
J
= 6.6 Hz, 1C), 55.7
(1C), 18.4 (d, 1JPC = 140.5 Hz, 1C), 16.2 (d, 3JPC = 5.1 Hz, 1C), 15.5
(1C), 6.5 (1C). MS[ES+, Q-TOF]: M + H+ = 428.12 m/z; 2 M + H+ =
855.27 m/z.
Isopropyl 5-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-2-meth-
ylphenyl propylphosphonate (21b). 21b was prepared by the reaction
of phenol 15 and phosphonochloridate 8b. It was purified by column
chromatography in 100% EtOAc. 21b was crystallized from
diisopropylether to give a white powder (0.118 g, 52%). 31P NMR
(DMSO-d6, 162 MHz): δ 28.5 ppm. 1H NMR (DMSO-d6, 400 MHz): δ
9.71 (s, 1H), 8.67 (d, 5JHH = 1.0 Hz, 1H), 7.95 (dd, 3JHH = 7.7 Hz, 4JHH
=
1.8 Hz, 1H), 7.77 (s, 1H), 7.44−7.48 (m, 2H), 7.45 (d, 5JHH = 1.2 Hz,
1H), 7.21 (d, 3JHH = 7.8 Hz, 1H), 7.18 (d, 3JHH = 7.6 Hz, 1H), 7.09 (t,
3JHH = 6.7 Hz, 1H), 4.65−4.75 (m, 1H), 3.91 (s, 3H), 2.19 (s, 3H),
1.88−1.99 (m, 2H), 1.56−1.71 (m, 2H), 1.28 (d, 3JHH = 6.1 Hz, 3H),
1.18 (d, 3JHH = 6.1 Hz, 3H), 1.02 (dt, 3JHH = 7.4 Hz, 4JPH = 1.4 Hz, 3H).
13C NMR (DMSO-d6, 100 MHz): δ 160.2 (1C), 159.4 (1C), 157.6
(1C), 157.5 (1C), 148.6 (d, 2JPC = 8.6 Hz, 1C), 138.8 (d, 4JPC = 1.2 Hz,
1C), 131.2 (1C), 131.0 (1C), 130.1 (1C), 125.7 (1C), 122.3 (d, 3JPC
=
5.5 Hz, 1C), 120.6 (1C), 115.6 (1C), 112.1 (1C), 111.6 (1C), 107.1
(1C), 70.5 (d, 2JPC = 6.7 Hz, 1C), 55.7 (1C), 27.8 (d, 1JPC = 139.7 Hz,
1C), 23.7 (d, 3JPC = 4.3 Hz, 1C), 23.5 (d, 3JPC = 4.3 Hz, 1C), 15.9 (d, 2JPC
3
= 5.5 Hz, 1C), 15.6 (1C), 14.9 (d, JPC = 17.2 Hz, 1C). MS[ES+, Q-
TOF]: M + H+ = 456.17 m/z; 2 M + H+ = 911.36 m/z.
Ethyl 5-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-2-methyl-
phenyl Phenylphosphonate (21c). 21c was prepared by the reaction
of phenol 15 and phosphonochloridate 8c. It was purified by column
chromatography in EtOAc:hexane (9:1). 21c was crystallized from
MeCN to give a white powder (0.021 g, 9%); mp = 146 °C (MeCN). 31P
NMR (DMSO-d6, 162 MHz): δ 14.7 ppm. 1H NMR (DMSO-d6, 400
MHz): δ 9.69 (s, 1H), 8.62 (s, 1H), 7.95 (dd, 3JHH = 7.5 Hz, 4JHH = 1.7
Hz, 1H), 7.83−7.89 (m, 2H), 7.76 (s, 1H), 7.67−7.73 (m, 1H), 7.58−
7.63 (m, 2H), 7.44−7.48 (m, 1H), 7.42 (s, 1H), 7.40−7.42 (m, 1H),
7.18 (d, 3JHH = 8.6 Hz, 2H), 7.09 (t, 3JHH = 7.3 Hz, 1H), 4.21 (m, 2H),
4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenol (19). It was
prepared according to the procedure used for compound 15 starting
from 4-amino-phenol 14c (0.600 g, 5.5 mmol) and 4-chloro-6-(2-
methoxyphenyl)-pyrimidine 13a (1.100 g, 5.0 mmol). 19 was
recrystallized from MeCN to give a brown powder (0.835 g, 57%);
mp = 249 °C (MeCN). 1H NMR (DMSO-d6, 300 MHz): δ 9.29 (s, 1H),
9.2 (s, 1H), 8.58 (s, 1H), 7.91 (d, 3JHH = 6.6 Hz, 1H), 7.38−7.5 (m, 3H),
7.29 (s, 1H), 7.15 (d, 3JHH = 8.3 Hz, 1H), 7.06 (t, 3JHH = 7.5 Hz, 1H),
3
3.90 (s, 3H), 2.15 (s, 3H), 1.29 (t, JHH = 7.0 Hz, 3H). MS[ES+, Q-
6.75 (d, JAABB AABB = 8.7 Hz, 2H), 3.86 (s, 3H). LC-MS: RtRt = 0.45 min;
J
1.85 min; 2.27 min. (ESI): m/z = 294 [M + H]+.
TOF]: M + H+ = 476.1 m/z; 2 M + H+ = 951.25 m/z.
4-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenol (20). It was
prepared according to the procedure used for compound 15 starting
from 4-amino-phenol 14c (0.600 g, 5.5 mmol) and 4-chloro-6-(3-
nitrophenyl)-pyrimidine 13b (1.178 g, 5.0 mmol). 20 was filtered from
Et2O to give a yellow powder (1.263 g, 82%); mp = 242−243 °C (Et2O).
1H NMR (DMSO-d6, 300 MHz): δ 9.49 (br, 2H), 8.79 (s, 1H), 8.66 (s,
1H), 8.41 (d, 3JHH = 6.3 Hz, 1H), 8.34 (d, 3JHH = 7.4 Hz, 1H), 7.82 (t,
3JHH = 7.5 Hz, 1H), 7.41 (d, JAABB = 7.0 Hz, 2H), 7.22 (s, 1H), 6.77 (d,
Ethyl 2-Methyl-5-{[6-(3-nitrophenyl)pyrimidin-4-yl]amino}phenyl
Ethylphosphonate (22a). 22a was prepared by the reaction of phenol
16 and phosphonochloridate 8a. 22a was recrystallized from MeCN to
give a pale-yellow powder (0.177 g, 79%); mp = 194−195 °C (MeCN).
1
31P NMR (DMSO-d6, 243 MHz): δ 29.8 ppm. H NMR (DMSO-d6,
300 MHz): δ 9.9 (s, 1H), 8.83 (s, 1H), 8.76 (s, 1H), 8.46 (d, 3JHH = 7.6
Hz, 1H), 8.36 (d, 3JHH = 7.9 Hz, 1H), 7.84 (t, 3JHH = 7.9 Hz, 1H), 7.74 (s,
1H), 7.46 (d, 3JHH = 7.9 Hz, 1H), 7.32 (s, 1H), 7.23 (d, 3JHH = 8.1 Hz,
1H), 4.07−4.19 (m, 2H), 2.21 (s, 3H), 1.91−2.07 (m, 2H), 1.25 (t, 3JHH
= 7.0 Hz, 3H), 1.13 (dt, 3JPH = 20.5 Hz, 3JHH = 7.6 Hz, 3H). LC-MS: Rt =
4.05 min. (ESI): m/z = 443 [M + H]+.
JAABB = 7.5 Hz, 2H). LC-MS: Rt = 2.85 min. (ESI): m/z = 309 [M + H]+.
General Procedure for the Preparation of Phosphonates (Ar-O-P
(O)ROR′). The appropriate phenol (15−20) (0.5 mmol) was dissolved
in anhydrous THF (25 mL) then KOtBu (0.067 g, 0.6 mmol) was added
in one portion. The mixture was stirred for 30 min at RT, and then the
corresponding phosphonochloridate 8a−c (0.75 mmol) was added. The
reaction mixture was stirred overnight at RT and concentrated under
vacuum. The residue was suspended in 2 M aqueous NaOH (30 mL),
and it was extracted with EtOAc (3 × 70 mL). The combined organic
phases were washed with brine, dried with MgSO4, and evaporated. For
further purification and characterization see the individual compounds.
Ethyl 5-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-2-methyl-
phenyl Ethylphosphonate (21a). 21a was prepared by the reaction
of phenol 15 and phosphonochloridate 8a. It was purified by column
chromatography: 100% EtOAc to EtOAc:MeOH (1:1) in gradient. 21a
was crystallized from MeCN to give a white powder (0.062 g, 29%); mp
= 134−135 °C (MeCN). 31P NMR (162 MHz): DMSO-d6, δ 30.9 ppm.
1H NMR (DMSO-d6, 400 MHz): δ 9.76 (s, 1H), 8.68 (s, 1H), 7.95 (dd,
Isopropyl 2-Methyl-5-{[6-(3-nitrophenyl)pyrimidin-4-yl]amino}-
phenyl Propylphosphonate (22b). 22b was prepared by the reaction
of phenol 16 and phosphonochloridate 8b. 22b was recrystallized from
MeCN to give a pale-yellow powder (0.111 g, 47%); mp = 164−166 °C
1
(MeCN). 31P NMR (DMSO-d6, 243 MHz): δ 27.6 ppm. H NMR
(DMSO-d6, 300 MHz): δ 9.85 (s, 1H), 8.82 (s, 1H), 8.74 (s, 1H), 8.45
(d, 3JHH = 7.8 Hz, 1H), 8.35 (dd, 3JHH = 7.4 Hz, 4JHH = 1.4 Hz, 1H), 7.83
(t, 3JHH = 8.0 Hz, 1H), 7.74 (s, 1H), 7.44 (d, 3JHH = 8.1 Hz, 1H), 7.36 (s,
3
1H), 7.21 (d, JHH = 8.3 Hz, 1H), 4.65−4.72 (m, 1H), 2.19 (s, 3H),
1.86−1.98 (m, 2H), 1.58−1.66 (m, 2H), 1.27 (d, 3JHH = 6.1 Hz, 3H),
1.17 (d, 3JHH = 6.1 Hz, 3H), 1.01 (t, 3JHH = 7.1 Hz, 3H). LC-MS: Rt =
4.55 min. (ESI): m/z = 271 [M + H]+.
5-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}-2-methylphenyl
Ethyl Ethylphosphonate (23a). The nitro-compound 22a (0.049 g,
0.11 mmol) was dissolved in the mixture of MeOH:CH2Cl2 (20 mL:10
mL). Ammonium formate (0.069 g, 1.1 mmol) and 10% Pd/C (0.005 g,
10% w/w) were added. Reaction mixture was stirred under inert
atmosphere overnight at RT. After the reduction was completed, catalyst
was filtered off on a celite pad and washed with MeOH (10 mL). Filtrate
was concentrated under vacuum. The residue was suspended in satd
aqueous Na2CO3 (30 mL) and extracted with EtOAc (3 × 70 mL). The
combined organic phases were washed with brine and dried with
MgSO4. After evaporation, the product 23a was crystallized from the
3JHH = 7.7 Hz, 4JHH = 1.7 Hz, 1H), 7.75 (s, 1H), 7.43−7.51 (m, 3H), 7.21
(d, 3JHH = 8.0 Hz, 1H), 7.19 (d, 3JHH = 8.0 Hz), 7.08 (t, 3JHH = 7.9 Hz,
1H), 4.07−4.20 (m, 2H), 3.9 (s, 3H), 2.2 (s, 3H), 1.97 (dq, 2JPH = 17.8
Hz, 3JHH = 7.6 Hz, 2H), 1.25 (t, 3JHH = 7.0 Hz, 3H), 1.16 (dt, 3JPH = 20.6
Hz, 3JHH = 7.6 Hz, 3H). 13C NMR (DMSO-d6, 100 MHz): δ 160.2 (1C),
159.4 (1C), 157.7 (1C), 157.5 (1C), 148.5 (d, 2JPC = 9.5 Hz, 1C), 138.8
(d, 4JPC = 1.5 Hz, 1C), 131.2 (1C), 131.0 (1C), 130.1 (1C), 125.7 (1C),
122.3 (d, 3JPC = 4.4 Hz, 1C), 120.6 (1C), 115.7 (1C), 112.1 (1C), 111.4
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mixture of 2-propanol and hexane to obtain an off-white powder (0.031
g, 67%); mp = 148−150 °C (i-PrOH, hexane). 31P NMR (DMSO-d6,
20.5 Hz, 3JHH = 7.6 Hz, 3H). LC-MS: Rt = 3.79 min. (ESI): m/z = 429
[M + H]+.
1
243 MHz): δ 29.9 ppm. H NMR (DMSO-d6, 300 MHz): δ 9.66 (s,
Isopropyl 3-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl Pro-
pylphosphonate (25b). 25b was prepared by the general method used
for phosphonates by the reaction of phenol 18 (0.090 g; 0.29 mmol) and
the phosphonic acid chloride 8b (0.057 g; 0.44 mmol). The product was
recrystallized from MeCN to give a yellow powder (0.057 g, 60%); mp =
163−164 °C (MeCN). 31P NMR (DMSO-d6, 243 MHz): δ 27.4 ppm.
1H NMR (DMSO-d6, 300 MHz): δ 10.04 (s, 1H), 8.84 (s, 1H), 8.79 (s,
1H), 8.63 (s, 1H), 7.70 (s, 1H), 7.42 (d, 3JHH = 7.6 Hz, 1H), 7.28 (s, 1H),
7.19 (d, 3JHH = 8.4 Hz, 1H), 7.00−7.20 (m, 3H), 6.60−6.70 (m, 1H),
5.24 (br, 2H), 4.11 (q, 3JHH = 6.7 Hz, 2H), 2.18 (s, 3H), 1.89−2.19 (m,
2H), 1.23 (t, 3JHH = 6.7 Hz, 3H), 1.14 (dt, 3JPH = 20.4 Hz, 3JHH = 7.4 Hz,
3H). LC-MS: Rt = 2.69 min. (ESI): m/z = 413 [M + H]+.
5-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}-2-methylphenyl Iso-
propyl Propylphosphonate (23b). The nitro-compound 22b (0.094 g,
0.2 mmol) was reduced with the method used for 23a. After evaporation
of organic phases, the product was dissolved in dry EtOAc (5 mL) and
the HCl salt was prepared with 0.5 mL of EtOAc satd with HCl. The
precipitate was filtered and washed with Et2O, affording a yellow powder
(0.060 g, 63%); mp = 165 °C dec (EtOAc). 31P NMR (DMSO-d6, 243
MHz): δ 27.7 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 11.00 (s, 1H),
8.82 (s, 1H), 7.73 (br, 2H), 7.20−7.60 (m, 7H), 6.00 (br, 2H), 4.65−
4.70 (m, 1H), 2.22 (s, 3H), 1.89−2.21 (m, 2H), 1.60−1.65 (m, 2H),
1H), 8.47 (d, 3JHH = 7.5 Hz, 1H), 8.37 (d, 3JHH = 8.1 Hz, 1H), 7.85 (t,
3JHH = 8.0 Hz, 1H), 7.76 (s, 1H), 7.53 (d, 3JHH = 8.1 Hz, 1H),7.44 (s,
1H), 7.34 (t, 3JHH = 8.1 Hz, 1H), 6.86 (d, 3JHH = 7.8 Hz, 1H), 4.68−4.75
(m, 1H), 1.84−1.96 (m, 2H), 1.57−1.65 (m, 2H), 1.28 (d, 3JHH = 6.0
Hz, 3H), 1.22 (d, 3JHH = 6.0 Hz, 3H), 1.00 (t, 3JHH = 7.1 Hz, 3H). LC-
MS: Rt = 4.28 min. (ESI): m/z = 457 [M + H]+.
Ethyl 3-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl Phenyl-
phosphonate (25c). 25c was prepared by the general method used for
phosphonates by the reaction of phenol 18 (0.102 g; 0.33 mmol) and
the phosphonochloridate 8c (0.102 g; 0.54 mmol). The product was
recrystallized from MeCN to give a cream-colored powder (0.083 g,
53%); mp = 162−164 °C (MeCN). 31P NMR (DMSO-d6, 243 MHz): δ
17.2 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 9.94 (s, 1H), 8.83 (s, 1H),
8.77 (s, 1H), 8.42 (dd, 3JPH = 27.2 Hz, 3JHH = 7.1 Hz, 2H), 7.80−7.90 (m,
3H), 7.76 (s, 1H), 7.65−7.7 (m, 1H), 7.55−7.65 (m, 2H), 7.47 (d, 3JHH
1.27 (d, 3JHH = 5.6 Hz, 3H), 1.18 (d, 3JHH = 5.6 Hz, 3H) 1.14 (t, 3JHH
6.8 Hz, 3H). LC-MS: Rt = 3.08 min. (ESI): m/z = 441 [M + H]+.
=
Ethyl 3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl Ethyl-
phosphonate (24a). 24a was prepared by the general method used for
phosphonates by the reaction of phenol 17 (0.060 g; 0.2 mmol) and the
phosphonochloridate 8a (0.047 g; 0.3 mmol). The product was purified
by preparative TLC in 100% EtOAc, giving a light-brown viscous oil
(0.049 g, 59%). 31P NMR (DMSO-d6, 243 MHz): δ 33.0 ppm. 1H NMR
(DMSO-d6, 300 MHz): δ 9.77 (s, 1H), 8.72 (s, 1H), 7.96 (dd, 3JHH = 7.7
Hz, 4JHH = 1.7 Hz, 1H), 7.73 (s, 1H), 7.53 (d, 3JHH = 8.3 Hz, 1H), 7.48 (s,
1H), 7.47 (dt, 3JHH = 8.4 Hz, 4JHH = 1.8 Hz, 1H), 7.32 (t, 3JHH = 8.3 Hz,
1H), 7.19 (d, 3JHH = 8.3 Hz, 1H), 7.08 (t, 3JHH = 7.4 Hz, 1H), 6.83 (d,
3JHH = 8.1 Hz, 1H), 4.09−4.20 (m, 2H), 3.91 (s, 3H), 1.86−2.00 (m,
= 7.2 Hz, 1H), 7.38 (s, 1H), 7.31 (t, 3JHH = 7.5 Hz, 1H), 6.82 (d, 3JHH
=
7.4 Hz, 1H), 4.18−4.24 (m, 2H), 1.29 (t, 3JHH = 6.2 Hz, 3H). LC-MS: Rt
= 4.28 min. (ESI): m/z = 457 [M + H]+.
3-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl Ethyl Ethyl-
phosphonate (26a). The nitro compound 25a (0.055 g, 0.13 mmol)
and SnCl2 (0.099 g; 0.52 mmol) were dissolved in EtOH (10 mL), and
the mixture was refluxed for 4 h. After the reaction was completed,
volatiles were evaporated then the residue was suspended in 2 M
aqueous NaOH (10 mL). The precipitate was filtered and washed with
EtOAc. The filtrate was extracted with EtOAc (3 × 20 mL), and the
organic phases were washed with brine and dried with MgSO4. After
evaporation, the pure product was dissolved in the minimal quantity of
EtOH, and the HCl salt was prepared by adding 4 M HCl in dioxane.
Volatiles were evaporated, and dry toluene (30 mL) was added and
distilled from the residue. The hydrochloride salt of the 26a was
suspended in Et2O, filtered, and dried to obtain a yellow powder (0.042
g, 74%); mp = 205−206 °C (EtOH, Et2O). 31P NMR (DMSO-d6, 243
MHz): δ 33.1 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 10.94 (s, 1H),
8.85 (s, 1H), 7.77 (s, 1H), 7.73 (br, 2H), 7.55−7.62 (m, 2H), 7.36−7.42
(m, 2H), 7.39 (s, 1H), 6.96 (d, 3JHH = 8.1 Hz, 1H), 5.50 (br, 2H), 4.05−
4.21 (m, 2H), 1.90−1.99 (m, 2H), 1.26 (t, 3JHH = 7.0 Hz, 3H), 1.13 (dt,
3JPH = 20.5 Hz, 3JHH = 7.7 Hz, 3H). LC-MS: Rt = 0.43 min; 2.48 min;
2H), 1.26 (t, 3JHH = 7.0 Hz, 3H), 1.13 (dt, 3JPH = 20.4 Hz, 3JHH = 7.6 Hz,
3H). LC-MS: Rt = 2.77 min. (ESI): m/z = 414 [M + H]+.
Isopropyl 3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl
Propylphosphonate (24b). 24b was prepared by the general method
used for phosphonates by the reaction of phenol 17 (0.060 g; 0.2 mmol)
and the phosphonochloridate 8b (0.055 g; 0.3 mmol). The product was
purified by preparative TLC in 100% EtOAc, affording a light-brown
viscous oil (0.058 g, 66%). 31P NMR (DMSO-d6, 243 MHz): δ 30.3
ppm. 1H NMR (DMSO-d6, 300 MHz): δ 9.76 (s, 1H), 8.71 (s, 1H), 7.96
(dd, 3JHH = 7.7 Hz, 4JHH = 1.6 Hz, 1H), 7.73 (s, 1H), 7.43−7.54 (m, 2H),
7.48 (s, 1H), 7.32 (t, 3JHH = 8.2 Hz, 1H), 7.19 (d, 3JHH = 8.3 Hz, 1H),
7.08 (t, 3JHH = 7.4 Hz, 1H), 6.83 (d, 3JHH = 8.0 Hz, 1H), 4.65−4.77 (m,
1H), 3.91 (s, 3H), 1.83−1.93 (m, 2H), 1.54−1.67 (m, 2H), 1.28 (d, 3JHH
= 6.2 Hz, 3H), 1.22 (d, 3JHH = 6.2 Hz, 3H), 1.00 (t, 3JHH = 7.2 Hz, 4JPH
1.0 Hz, 3H). LC-MS: Rt = 3.16 min. (ESI): m/z = 442 [M + H]+.
=
2.57 min. (ESI): m/z = 399 [M + H]+.
Ethyl 3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl Phe-
nylphosphonate (24c). 24c was prepared by the general method used
for phosphonates by the reaction of phenol 17 (0.060 g; 0.2 mmol) and
the phosphonochloridate 8c (0.061 g; 0.3 mmol). The product was
purified by preparative TLC in EtOAc:hexane = 1:1, giving a light-brown
viscous oil (0.037 g, 40%). 31P NMR (DMSO-d6, 243 MHz): δ 17.1
ppm. 1H NMR (DMSO-d6, 300 MHz): δ 9.75 (s, 1H), 8.68 (s, 1H), 7.95
3-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl Isopropyl
Propylphosphonate (26b). Compound 25b (0.133 g, 0.29 mmol)
was reduced according to the preparation of 26a, giving a green−yellow
powder (0.109 g, 81%); mp = 148−150 °C (EtOH, Et2O). 31P NMR
(DMSO-d6, 243 MHz): δ 30.4 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
10.35 (s, 1H), 8.72 (s, 1H), 7.70 (s, 1H), 7.30−7.60 (m, 3H), 7.20−7.30
(m, 3H), 7.06 (br, 1H), 6.84 (br, 1H), 4.67 (br, 1H), 3.70 (br, 2H2),
1.75−1.90 (m, 2H), 1.50−1.60 (m, 2H), 1.24 (s, 3H), 1.19 (s, 3H), 0.96
(br, 3H). LC-MS: Rt = 3.00 min. (ESI): m/z = 427 [M + H]+.
3-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl Ethyl Phenyl-
phosphonate (26c). Compound 25c (0.065 g, 0.14 mmol) was reduced
according to the preparation of 26a, giving a yellow powder (0.051 g,
76%); mp = 214−215 °C (EtOH, Et2O). 31P NMR (DMSO-d6, 243
MHz): δ 17.3 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 10.89 (s, 1H),
(dd, 3JHH = 7.5 Hz, 4JHH = 1.3 Hz, 1H), 7.84 (dd, 3JPH = 13.5 Hz, 3JHH
=
7.2 Hz; 2H), 7.75 (s, 1H) ; 7.65−7.71 (m, 1H), 7.55−7.62 (m, 2H),
7.4−7.5 (m, 2H), 7.45 (s, 1H), 7.28 (t, 3JHH = 8.1 Hz, 1H), 7.18 (d, 3JHH
= 8.1 Hz, 1H), 7.08 (t, 3JHH = 7.3 Hz, 1H), 6.79 (d, 3JHH = 8.1 Hz, 1H),
4.15−4.25 (m, 2H), 3.90 (s, 3H), 1.29 (t, 3JHH = 7.2 Hz, 3H). LC-MS: Rt
= 3.14 min. (ESI): m/z = 462 [M + H]+.
Ethyl 3-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl Ethyl-
phosphonate (25a). 25a was prepared by the general method used
for phosphonates by the reaction of phenol 18 (0.090 g; 0.29 mmol) and
the phosphonochloridate 8a (0.068 g; 0.44 mmol). The product was
recrystallized from MeCN to give a yellow powder (0.075 g, 60%); mp =
142−144 °C (MeCN). 31P NMR (DMSO-d6, 243 MHz): δ 33.0 ppm.
1H NMR (DMSO-d6, 300 MHz): δ 9.96 (s, 1H), 8.83 (s, 1H), 8.80 (s,
8.82 (s, 1H), 7.50−7.90 (m, 10H), 7.30−7.50 (m, 3H), 6.91 (d, 3JHH
=
7.8 Hz, 1H), 5.40 (br, 2H), 4.15−4.26 (m, 2H), 1.29 (t, 3JHH = 6.9 Hz,
3H). LC-MS: Rt = 2.96 min. (ESI): m/z = 447 [M + H]+.
Ethyl 4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl Ethyl-
phosphonate (27a). 27a was prepared by the general method used for
phosphonates by the reaction of phenol 19 (0.033 g; 0.11 mmol) and
the phosphonochloridate 8a (0.060 g; 0.17 mmol). The product was
purified by preparative TLC in 100% EtOAc, providing a light-brown
viscous oil (0.022 g 48%). 1H NMR (DMSO-d6, 300 MHz): δ 9.64 (s,
1H), 8.47 (d, 3JHH = 7.7 Hz, 1H), 8.37 (d, 3JHH = 8.0 Hz, 1H), 7.85 (t,
3JHH = 8.0 Hz, 1H), 7.75 (s, 1H), 7.53 (d, 3JHH = 7.7. Hz, 1H), 7.41 (s,
1H), 7.35 (t, 3JHH = 8.1 Hz, 1H), 6.87 (d, 3JHH = 7.7 Hz, 1H), 4.08−4.2
(m, 2H), 1.87−2.01 (m, 2H), 1.26 (t, 3JHH = 7.0 Hz, 3H), 1.13 (dt, 3JPH
=
3952
dx.doi.org/10.1021/jm401742r | J. Med. Chem. 2014, 57, 3939−3965

Journal of Medicinal Chemistry
Article
1H), 8.67 (s, 1H), 7. 95 (dd, 3JHH = 7.6 Hz, 4JHH = 1.5 Hz, 1H), 7.69 (d,
JAABB = 8.8 Hz, 2H), 7.45 (dt, 3JHH = 8.6 Hz, 4JHH = 1.5 Hz, 1H), 7.42 (s,
1H), 7.18 (d, JAABB = 8.3 Hz, 2H), 7.17 (d, 3JHH = 7.8 Hz, 1H), 7.08 (t,
3JHH = 7.5 Hz, 1H), 4.04−4.16 (m, 2H), 3.90 (s, 3H), 1.83−1.98 (m,
7.12−7.23 (m, 4H), 7.09 (s, 1H, H12), 6.68 (d, 3JHH = 5.6 Hz, 1H), 5.26
(br, 2H), 4.64−4.71 (m, 1H), 1.81−1.9 (m, 2H), 1.58−1.63 (m, 2H),
1.27 (d, 3JHH = 6.2 Hz, 3H), 1.20 (d, 3JHH = 6.2 Hz, 3H), 1.00 (t, 3JHH
6.3 Hz, 3H). LC-MS: Rt = 2.78 min. (ESI): m/z = 427 [M + H]+.
=
2H), 1.24 (t, 3JHH = 7.0 Hz, 3H), 1.11 (dt, 3JPH = 20.3 Hz, 3JHH = 7.6 Hz,
3H). LC-MS: Rt = 2.70 min. (ESI): m/z = 414 [M + H]+.
4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl Hydrogen
Ethylphosphonate (30a). Chlorotrimethylsilane (0.21 mL, 1.65
mmol) and potassium iodide (0.274 g; 1.65 mmol) were dissolved in
anhydrous acetone (20 mL). The mixture was stirred for 30 min at RT
then the solution of phosphonate 21a (0.235 g, 0.55 mmol in 30 mL
anhydrous acetone) was added. The reaction mixture was refluxed for 20
h, and then it was concentrated under vacuum. The crude product was
suspended in satd NH4Cl solution and 0.5 mL of EtOAc. The
suspension was stirred for 1 h at RT then it was filtered off and washed
with Et2O, giving 30a as a yellow powder was prepared (0.154 g, 70%);
mp = 240−242 °C (water). 31P NMR (DMSO-d6, 243 MHz): δ 26.9
ppm. 1H NMR (DMSO-d6, at 110 °C, 300 MHz): δ 10.02 (br, 1H), 8.71
(s, 1H), 7.85 (d, 3JHH = 7.8 Hz, 1H), 7.61 (s, 1H), 7.40−7.55 (m, 3H),
7.41 (s, 1H), 7.2 (d, 3JHH = 7.4 Hz, 1H), 7.11 (t, 3JHH = 7.4 Hz, 1H), 4.89
Isopropyl 4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl
Isopropylphosphonate (27b). 27b was prepared by the general method
used for phosphonates by the reaction of phenol 19 (0.100 g; 0.34
mmol) and the phosphonochloridate 8b (0.094 g; 0.51 mmol). The
product was purified by preparative TLC in 100% EtOAc, yielding a
yellow powder (0.020 g, 13%); mp = 119−121 °C (CH2Cl2). 31P NMR
(DMSO-d6, 243 MHz): δ 30.6 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.63 (s, 1H), 8.67 (s, 1H), 7.95 (d, 3JHH = 6.8 Hz, 1H), 7.69 (JAABB = 7.4
Hz, 2H), 7.35−7.50 (m, 2H), 7.15−7.18 (m, 3H), 7.05−7.13 (m, 1H),
4.60−4.70 (m, 1H), 3.9 (s, 3H), 1.81−1.90 (m, 2H), 1.49−1.70 (m,
2H), 1.27 (d, 3JHH = 5.1 Hz, 3H), 1.20 (d, 3JHH = 5.1 Hz, 3H), 0.99 (t,
3JHH = 6.6 Hz, 3H). LC-MS: Rt = 3.07 min. (ESI): m/z = 442 [M + H]+.
(br, 1H), 3.91 (s, 3H), 2.24 (s, 3H), 1.77−1.92 (m, 2H), 1.17 (dt, 3JPH
=
Ethyl 4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl Phe-
nylphosphonate (27c). 27c was prepared by the general method used
for phosphonates by the reaction of phenol 19 (0.033 g; 0.11 mmol) and
the phosphonochloridate 8c (0.035 g; 0.17 mmol). The product was
purified by preparative TLC in EtOAc:hexane = 1:1, affording a light-
brown viscous oil (0.041 g, 81%). 1H NMR (DMSO-d6, 300 MHz): δ
9.63 (s, 1H), 8.66 (s, 1H), 7.94 (dd, 3JHH = 7.7 Hz, 4JHH = 1.2 Hz, 1H),
7.82 (dd, 3JPH = 13.3 Hz, 3JHH = 7.1 Hz, 2H), 7.60−7.75 (m, 3H), 7.50−
19.9 Hz, 3JHH = 7.6 Hz, 3H). LC-MS: Rt = 2.43 min; (ESI): m/z = 400
[M + H]+. MS[ES+, Q-TOF]: M + H+ = 400.11 m/z; 2 M + H+ = 799.18
m/z.
4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl Hydrogen
Propylphosphonate (30b). Deprotection of phosphonate 21b (0.050
g; 0.11 mmol) was performed according to the preparation of 30a,
affording 30b as a brown powder (0.036 g, 79%); mp = 170 °C (water).
1
31P NMR (DMSO-d6, 162 MHz): δ 26.6 ppm. H NMR (DMSO-d6,
7.60 (m, 2H), 7.45 (t, 3JHH = 7.8 Hz, 1H), 7.40 (s, 1H), 7.17 (d, 3JHH
=
400 MHz): δ 10.48 (br, 1H), 8.81 (s, 1H), 7.78 (d, 3JHH = 7.4 Hz, 1H),
7.57 (s, 1H), 7.56 (t, 3JHH = 7.5 Hz, 1H), 7.49 (d, 3JHH = 8.2 Hz, 1H),
7.32 (s, 1H), 7.25 (d, 3JHH = 8.4 Hz, 1H), 7.24 (d, 3JHH = 8.6 Hz, 1H),
8.3 Hz, 1H), 7.13 (d, JAABB = 8.5 Hz, 2H), 7.07 (t, 3JHH = 7.6 Hz, 1H),
4.13−4.22 (m, 2H), 3.88 (s, 3H), 1.27 (t, 3JHH = 7.0 Hz, 3H). LC-MS: Rt
= 3.06 min. (ESI): m/z = 462 [M + H]+.
3
7.14 (t, JHH = 7.5 Hz, 1H), 3.50 (s, 1H), 3.90 (s, 3H), 2.21 (s, 3H),
Ethyl 4-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl Ethyl-
phosphonate (28a). 28a was prepared by the general method used
for phosphonates by the reaction of phenol 20 (0.150 g; 0.49 mmol) and
the phosphonochloridate 8a (0.115 g; 0.74 mmol). After recrystalliza-
tion from MeCN, a yellow powder was obtained (0.085 g, 41%); mp =
1.77−1.86 (m, 2H), 1.58−1.67 (m, 2H), 1.01 (t, 3JHH = 7.1 Hz, 3H). LC-
MS: Rt = 2.53 min. (ESI): m/z = 414 [M + H]+. MS[ES+, Q-TOF]: M +
H+ = 414.12 m/z; 2 M + H+ = 827.22 m/z.
Diethyl [Hydroxy(3-nitrophenyl)methyl]phosphonate (31). 3-
Nitrobenzaldehyde (7.55 g, 50 mmol) was dissolved in toluene (50
mL) then diethyl phosphite (7.59 g, 55 mmol) and triethylamine (5.56
g, 55 mmol) were added and stirred at RT overnight. Water (50 mL) was
added to the reaction mixture then the organic phase was washed with 1
M aqueous HCl solution (50 mL). Combined aqueous phases were
extracted with toluene (30 mL). The organic phases were washed with
brine and dried with MgSO4. After concentration, 31 was recrystallized
from toluene to give a pale-yellow powder (11.04 g, 76%); mp = 92 °C
1
187 °C (MeCN). 31P NMR (DMSO-d6, 243 MHz): δ 33.2 ppm. H
NMR (DMSO-d6, 300 MHz): δ 9.81 (s, 1H), 8.86 (s, 1H), 8.75 (s, 1H),
8.45 (d, 3JHH = 7.8 Hz, 1H), 8.36 (d, 3JHH = 8.1 Hz, 1H), 7.84 (t, 3JHH
8.0 Hz, 1H), 7.71 (d, JAABB = 8.8 Hz, 2H), 7.34 (s, 1H), 7.19 (d, JAABB
=
=
8.5 Hz, 2H), 4.04−4.19 (m, 2H), 1.90 (dq, 3JPH = 7.6 Hz, 3JHH = 7.6 Hz,
2H), 1.25 (t, 3JHH = 7.0 Hz, 3H), 1.12 (dt, 3JPH = 20.4 Hz, 3JHH = 7.6 Hz,
3H). LC-MS: Rt = 3.63 min. (ESI): m/z = 429 [M + H]+.
Isopropyl 4-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl Pro-
pylphosphonate (28b). 28b was prepared by the general method used
for phosphonates by the reaction of phenol 20 (0.154 g; 0.50 mmol) and
the phosphonochloridate 8b (0.138 g; 0.75 mmol). After recrystalliza-
tion from MeCN, a cream-colored powder was obtained (0.012 g, 12%);
mp = 187 °C (MeCN). 31P NMR (DMSO-d6, 243 MHz): δ 27.6 ppm.
1H NMR (DMSO-d6, 300 MHz): δ 9.86 (s, 1H), 8.82 (s, 1H), 8.75 (s,
1
(toluene). 31P NMR (DMSO-d6, 242 MHz): δ 19.8 ppm. H NMR
(DMSO-d6, 300 MHz): δ 8.28 (d, 4JHH = 1.6 Hz, 1H), 8.14 (d, 3JHH = 8.0
Hz, 1H), 7.86 (d, 3JHH = 7.5 Hz, 1H), 7.65 (t, 3JHH = 7.9 Hz, 1H), 6.52
(dd, 3JPH = 14.5 Hz, 3JHH = 5.9 Hz, 1H), 5.2 (dd, 2JPH = 14.1 Hz, 3JHH
=
5.9 Hz, 1H), 3.90−4.04 (m, 4H), 1.16 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt
= 2.83 min. (ESI): m/z = 290 [M + H]+.
1H), 8.45 (d, 3JHH = 7.3 Hz, 1H), 8.36 (d, 3JHH = 7.6 Hz, 1H), 7.84 (t,
Diethyl [Hydroxy(4-nitrophenyl)methyl]phosphonate (32). 32 was
prepared similarly to compound 31 from 4-nitrobenzaldehyde (7.550 g,
50 mmol). 32 was prepared as a pale-yellow powder (8.67 g, 60%); mp =
88−90 °C (toluene). 31P NMR (DMSO-d6, 242 MHz): δ 19.4 ppm. 1H
NMR (DMSO-d6, 300 MHz): δ 8.21 (d, JAABB = 8.6 Hz, 2H), 7.68 (dd,
JAABB = 8.9 Hz, J = 2.2 Hz 2H), 6.50 (dd, 4JPH = 14.6 Hz, 3JHH = 5.7 Hz,
1H), 5.18 (dd, 2JPH = 15.4 Hz, 3JHH = 5.7 Hz), 3.90−4.05 (m, 4H), 1.16
(dt, 3JHH = 7.0 Hz, J = 3.0 Hz, 6H). LC-MS: Rt = 2.84 min. (ESI): m/z =
290 [M + H]+.
3JHH = 8.0 Hz, 1H), 7.71 (d, JAABB = 8.1 Hz, 2H), 7.36 (s, 1H), 7.18 (d,
JAABB = 8.1 Hz, 2H), 4.60−4.70 (m, 1H), 1.81−1.93 (m, 2H), 1.55−1.63
(m, 2H), 1.27 (d, 3JHH = 5.8 Hz, 3H), 1.20 (d, 3JHH = 5.7 Hz, 3H), 1.00
(t, 3JHH = 6.6 Hz, 3H). LC-MS: Rt = 4.20 min. (ESI): m/z = 457 [M +
H]+.
4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl Ethyl Ethyl-
phosphonate (29a). Compound 28a (0.067 g, 0.16 mmol) was
reduced according to the preparation of 26a, giving a yellow powder
(0.043 g, 62%); mp = 207−209 °C (EtOH, Et2O). 1H NMR (DMSO-d6,
300 MHz): δ 9.63 (s, 1H), 8.64 (s, 1H), 7.68 (d, JAABB = 8.7 Hz, 2H),
7.28 (s, 1H), 7.09−7.19 (m, 5H), 6.68 (d, 3JHH = 6.4 Hz, 1H), 5.26 (br,
2H), 4.40−4.17 (m, 2H), 1.83−1.98 (m, 2H), 1.24 (t, 3JHH = 7.05 Hz,
3H), 1.11 (dt, 3JPH = 12.7 Hz, 3JHH = 7.6 Hz, 3H). LC-MS: Rt = 0.43 min
2.28 min; 2.47 min. (ESI): m/z = 399 [M + H]+.
4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl Isopropyl
Propylphosphonate (29b). Compound 28b (0.076 g, 0.17 mmol)
was reduced according to the preparation of 26a, affording a yellow
powder (0.016 g, 21%); mp = 150−160 °C (EtOH, Et2O). 31P NMR
(DMSO-d6, 243 MHz): δ 27.6 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.62 (s, 1H), 8.64 (s, 1H), 7.68 (d, JAABB = 8.9 Hz, 2H), 7.28 (s, 1H),
Diethyl [(3-Aminophenyl)(hydroxy)methyl]phosphonate (33). Di-
ethyl [hydroxy(3-nitrophenyl)methyl]phosphonate 31 (1.455 g; 5.0
mmol) and ammonium formate (3.150 g, 50.0 mmol) were dissolved in
MeOH (100 mL) under un inert atmosphere. Then 10 m/m % Pd/C
(0.145 g, 10 w/w%) was added to the mixture and stirred for 24 h at RT.
Catalyst was filtered off on a Celite pad and washed with MeOH (20
mL). After concentration of the filtrate, the residue was suspended in
satd aqueous Na2CO3 (50 mL) and it was extracted with EtOAc (3 × 50
mL). The combined organics were washed with brine and dried with
MgSO4. After evaporation of volatiles under reduced pressure, 33 was
obtained as a yellow oil (0.936 g; 72%). 31P NMR (DMSO-d6, 242
MHz): δ 21.1 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 6.93 (t, 3JHH
=
3953
dx.doi.org/10.1021/jm401742r | J. Med. Chem. 2014, 57, 3939−3965

Journal of Medicinal Chemistry
Article
7.7 Hz, 1H), 6.65 (d, 4JHH = 1.5 Hz, 1H), 6.54 (d, 3JHH = 7.3 Hz, 1H),
6.44 (d, 3JHH = 7.8 Hz, 1H), 5.92 (dd, 3JPH = 16.7 Hz, 3JHH = 5.5 Hz, 1H),
5.00 (br, 2H), 4.7 (dd, 2JPH = 13.1 Hz, 3JHH = 5.5 Hz, 1H), 3.82−3.99 (m,
4H), 1.15 (dt, 3JHH = 7.0 Hz, 4JPH = 10.6 Hz, 6H). LC-MS: Rt = 0.48 min;
1.49 min; 2.18 min. (ESI): m/z = 260 [M +H]+.
Diethyl [(4-Aminophenyl)(hydroxy)methyl]phosphonate (34). 34
was synthesized according to the preparation of compound 33 from
diethyl [hydroxy(4-nitrophenyl)methyl]phosphonate 32 (1.50 g, 5.19
mmol). 34 was filtered from Et2O to give a yellow powder (1.16 g, 86%);
mp = 157−158 °C (Et2O). 31P NMR (DMSO-d6, 242 MHz): δ 21.8
ppm. 1H NMR (DMSO-d6, 300 MHz): δ 7.07 (dd, JAABB = 8.3 Hz, J = 1.7
Hz 2H), 6.50 (d, JAABB = 8.3 Hz, 2H), 5.80 (br, 1H), 5.03 (br, 2H), 4.66
(d, 2JPH = 11.7 Hz), 3.90−4.01 (m, 3JHH = 7.2 Hz, 2H), 3.70−3.90 (m,
3JHH = 7.2 Hz, 2H), 1.18 (t, 3JHH = 7.0 Hz, 3H), 1.11 (t, 3JHH = 7.0 Hz,
3H). LC-MS: decomposition, Rt = 0.45 min. (ESI): m/z = 242 [M −
H2O + H]+.
MgSO4. After concentration, the ethyl benzoate was obtained as a pale-
yellow liquid which crystallizes to yellow crystals upon standing (4.978
g, 92%). 1H NMR (CDCl3, 400 MHz): δ 8.75 (d, 4JHH = 2.5 Hz, 1H),
8.23 (dd, 3JHH = 8.3 Hz, 4JHH = 2.5 Hz, 1H), 7.43 (d, 3JHH = 8.4 Hz, 1H),
4.42 (q, 3JHH = 7.4 Hz, 2H), 2.72 (s, 3H), 1.44 (t, 3JHH = 7.1 Hz, 3H).
LC-MS: Rt = 4.11 min. (ESI): m/z = no molecular ion was observed.
The ethyl 2-methyl-5-nitrobenzoate (4.967 g; 23.8 mmol) was dissolved
in dry THF (100 mL) and cooled to 0 °C. LiAlH4 (3.612 g, 95.1 mmol)
was added in small portions. After the addition, the reaction mixture was
stirred for 2.5 h at RT. Then 2-propanol was added drop by drop until
the release of hydrogen stopped. After the addition of 2 M aqueous
NaOH was added (50 mL), the precipitated white gel was filtered on
celite and washed with EtOAc. The filtrate was separated, and the
aqueous layer was extracted with EtOAc (3 × 75 mL). The combined
organic phases were washed with brine, dried with MgSO4, and
concentrated. The benzyl alcohol intermediate was titurated and filtered
from Et2O to give an orange powder (1.97 g, 50%); mp = 159−162 °C
Diethyl (3-Aminobenzyl)phosphonate (35). Diethyl [hydroxy(3-
nitrophenyl)methyl]phosphonate 31 (1.455 g; 5.0 mmol) and triethyl-
amine (0.83 mL, 6.0 mmol) were dissolved in dry THF (25 mL) and
cooled to 0 °C. Methanesulfonic acid chloride (0.43 mL, 5.5 mmol) was
added dropwise, and the resulting mixture was stirred for 2 h at RT. After
concentration, the residue was partitioned between water and EtOAc
(50−50 mL). Aqueous phase was extracted with EtOAc (2 × 50 mL).
The combined organic phases were washed with 1 M aqueous HCl (50
mL), with satd aqueous NaHCO3 (50 mL) and with brine and dried
with MgSO4. After concentration, the methanesulfonate intermediate
was filtered from Et2O to give a white powder (1.343 g, 73%); mp = 81−
82 °C (Et2O). 31P NMR (DMSO-d6, 242 MHz): δ 13.2 ppm. 1H NMR
(DMSO-d6, 300 MHz): δ 8.33 (s, 1H), 8.25 (d, 3JHH = 8.0 Hz, 1H), 7.93
(d, 3JHH = 7.4 Hz, 1H), 7.75 (t, 3JHH = 8.0 Hz, 1H), 6.35 (d, 2JPH = 15.9
(Et2O). 1H NMR (CDCl3, 400 MHz): δ 7.87 (s, 1H), 7.71 (dd, 3JHH
=
8.1 Hz, 4JHH = 2.3 Hz, 1H), 7.25 (d, 3JHH = 8.1 Hz, 1H), 4.73 (s, 2H),
2.36 (s, 3H), 1.52 (br, 1H). LC-MS: Rt = 3.43 min. (ESI): m/z = no
molecular ion was observed. The (2-methyl-5-nitro-phenyl)-methanol
(0.20 g; 1.2 mmol) was dissolved in dry THF (20 mL) and cooled to
−10 °C. PBr3 (0.056 mL, 0.6 mmol) was added in one portion. After 1.5
h stirring at RT, the reaction mixture was concentrated to dryness. The
residue was dissolved in EtOAc:THF (2:1, 80 mL), and it was washed
with water (2 × 30 mL) and with brine. The organic layer was dried with
Na2SO4 than it was concentrated. The crude benzyl bromide was
titurated and filtered from Et2O to give a yellow to brown powder (0.233
1
g, 85%); mp = 195−198 °C (Et2O). H NMR (CDCl3, 400 MHz): δ
7.81 (d, 4JHH = 2.1 Hz, 1H), 7.72 (dd, 3JHH = 7.9 Hz, 4JHH = 2.1 Hz, 1H),
Hz), 3.96−4.14 (m, 4H), 3.22 (s, 3H), 1.18 (dt, 3JHH = 7.0 Hz, 4JPH
=
3
7.25 (d, JHH = 8.2 Hz, 1H), 4.52 (s, 2H), 2.43 (s, 3H). LCMS:
10.0 Hz, 6H). LC-MS: Rt = 3.33 min; (ESI): m/z = 368 [M + H]+.
(Diethoxyphosphoryl)(3-nitrophenyl)methyl methanesulfonate (0.367
g, 1.0 mmol) was dissolved in MeOH (20 mL) under an inert
atmosphere. Then 10 m/m % Pd/C (0.037 g, 10 w/w %) and 70%
aqueous solution of hydrazine (2 mL) were added carefully under
stirring. After 24 h vigorous stirring at RT, the catalyst was filtered off on
a celite pad and washed with MeOH (20 mL) then the filtrate was
concentrated under vacuum. The residue was suspended in sat aqueous
NaHCO3 (20 mL), and it was extracted with EtOAc (3 × 20 mL). The
organic phases were washed with brine and dried with MgSO4. After
concentration under vacuum, the title compound 35 was obtained as a
yellow oil (0.190 g, 78%). 31P NMR (DMSO-d6, 242 MHz): δ 25.5 ppm.
1H NMR (DMSO-d6, 300 MHz): δ 8.20 (br, 2H), 7.17 (t, 3JHH = 7.7 Hz,
1H), 6.81−6.88 (m, 3H), 3.93 (dq, 3JPH≈3JHH = 7.1 Hz, 4H), 3.14 (d,
2JPH = 21.6 Hz, 2H), 1.16 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt = 0.46 min;
1.89 min. (ESI): m/z = 244 [M + H]+.
compound was decomposed on LC. 2-(Bromomethyl)-1-methyl-4-
nitrobenzene (0.23 g, 1.0 mmol) was suspended in triethyl-phosphite (4
mL, 23.0 mmol) and was stirred in a closed flask in a microwave reactor
at 100 °C for 10 min. The mixture was cooled to 0 °C, and 36% aqueous
HCl solution (1 mL) was added. After 30 min stirring, the product was
extracted with EtOAc:THF (2:1, 3 × 20 mL). The combined organics
were washed with satd aqueous NaHCO3 (20 mL) and with brine and
dried with MgSO4. After evaporation, the benzyl-phosphonate was
crystallized from Et2O to give an orange powder (0.11 g, 38%); mp =
117−119 °C (Et2O). 1H NMR (DMSO-d6, 300 MHz): δ 7.77 (s, 1H),
7.67 (d, 3JHH = 8.0 Hz, 1H), 7.39 (d, 3JHH = 8.0 Hz, 1H), 3.90−4.03 (m,
4H), 3.40 (d, 2JPH = ∼22.0 Hz, 2H), 2.43 (s, 3H), 1.18 (t, 3JHH = 7.0 Hz,
6H). LC-MS: Rt = 4.18 min. (ESI): m/z = no molecular ion was
observed. Diethyl (2-methyl-5-nitrobenzyl)phosphonate (0.100 g, 0.36
mmol) was dissolved in MeOH (20 mL), and ammonium formate
(0.227 g, 3.60 mmol) was added to the mixture under inert atmosphere
followed by the addition of 10 m/m % Pd/C (0.010 g, 10 w/w%). The
reaction mixture was stirred at RT for 20 h, and the catalyst was filtered
off on a celite pad and washed with MeOH (20 mL) then the filtrate was
evaporated to dry. The residue was dissolved in EtOAc (50 mL) and was
washed with satd aqueous NaHCO3 (20 mL) with brine and dried with
MgSO4. After evaporation of the volatiles under reduced pressure the
title compound 37 was obtained as a yellow oil (0.071 g, 77%), which
was used without further purification. 1H NMR (DMSO-d6, 300 MHz):
δ 6.78 (d, 3JHH = 8.0 Hz, 1H), 6.45 (t, 4JHH = 2.5 Hz, 1H), 6.35 (dt, 3JHH
= 8.0 Hz, 4JHH = 2.3 Hz, 1H), 4.77 (br, 2H), 3.85−3.98 (m, 4H), 2.98 (d,
2JPH = 21.6 Hz, 2H), 2.13 (s, 3H), 1.17 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt
Diethyl (4-Aminobenzyl)phosphonate (36). Diethyl [hydroxy(4-
nitrophenyl)methyl]phosphonate 32 (1.445 g; 5.0 mmol) was
mesylated similarly to the synthesis of 35. The methanesulfonate
intermediate was isolated as pale-yellow powder (0.954 g, 52%); mp =
1
79−80 °C (Et2O). 31P NMR (DMSO-d6, 242 MHz): δ 12.9 ppm. H
NMR (DMSO-d6, 300 MHz): δ 8.28 (d, JAABB = 8.7 Hz, 2H), 7.74 (dd,
JAABB = 8.8 Hz, J = 2.0 Hz, 2H), 6.33 (d, 2JPH = 16.7 Hz, 1H), 3.96−4.12
(m, 4H), 3.23 (s, 3H, H8), 1.18 (dt, 3JHH = 7.0 Hz, J = 4.2 Hz, 6H). LC-
MS: Rt = 3.34 min. (ESI): m/z = 368 [M + H]+. (Diethoxyphosphoryl)-
(4-nitrophenyl)methyl methanesulfonate (0.734 g; 2.0 mmol) was
hydrogenated according to the preparation of compound 35. The title
compound 36 was obtained as a white powder (0.456 g, 94%); mp =
90−91 °C (EtOAc). 31P NMR (DMSO-d6, 242 MHz): δ 26.7 ppm. 1H
NMR (DMSO-d6, 300 MHz): δ 6.90 (dd, JAABB = 8.3 Hz, J = 2.2 Hz 2H),
6.49 (d, JAABB = 8.3 Hz, 2H), 4.92 (br, 2H), 3.90 (dq, 3JPH = 7.3 Hz, 3JHH
= 7.2 Hz, 4H), 2.97 (d, 2JPH = 20.6 Hz, 2H), 1.16 (t, 3JHH = 7.0 Hz, 6H).
LC-MS: Rt = 0.45 min; 1.69 min. (ESI): m/z = 244 [M + H]+.
= 0.45 min; 2.16 min. (ESI): m/z = 258 [M + H]+.
General Procedure for the Coupling Reaction of 4-Chloro-6-
phenyl-pyrimidines (13) and Phosphorus Containing Anilines. The
appropriate aniline was dissolved in the necessary volume of EtOH and a
few drops of 4 M HCl in dioxane were added (approximately 0.1 mL).
The volatiles were evaporated then dry toluene (30 mL) was distilled
from the residue. The 4-chloro-6-phenyl-pyrimidine 13 was added to
the residue then it was refluxed for 3−24 h in anhydrous 2-propanol (35
mL). Reactions were followed by TLC with toluene:MeOH (4:1) as
eluent. After the reaction was complete, volatiles were evaporated then
the residue was suspended in satd aqueous NaHCO3 (30 mL). The
product was extracted with EtOAc (3 × 70 mL). The combined organic
Diethyl (5-Amino-2-methylbenzyl)phosphonate (37). 2-Methyl-5-
nitro benzoic acid (5.00 g; 27.6 mmol) was dissolved in abs EtOH (200
mL), and 96% H2SO4 (2 mL) was added to the solution then it was
refluxed for 24 h. The reaction mixture was concentrated under vacuum,
and the residue was dissolved in EtOAc (200 mL). The organic phase
was washed with 1 M NaOH (2 × 50 mL) with brine and dried with
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phases were washed with brine and dried with MgSO4, and then solvents
were evaporated under reduced pressure. For the exact quantity of
reagents, for further purifications, yields, and analytical data see the
examples.
reacted. The crude product was recrystallized from MeCN to give a
yellow powder (0.212 g, 50%); mp = 151 °C (MeCN). 31P NMR
(DMSO-d6, 242 MHz): δ 25.9 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.57 (s, 1H; 8.66 (s, 1H), 7.93 (d, 3JHH = 7.5 Hz, 1H), 7.62 (d, JAABB = 8.2
Hz, 2H), 7.43 (t, 3JHH = 8.4 Hz, 1H), 7.42 (s, 1H), 7.22 (dd, JAABB = 8.4
Hz, 4JPH = 1.8 Hz, 2H), 7.16 (d, 3JHH = 8.3 Hz, 1H), 7.06 (t, 3JHH = 7.5
Hz, 1H), 3.90−3.99 (m, 4H), 3.88 (s, 3H, H20), 3.15 (d, 2JPH = 21.2 Hz,
2H), 1.17 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt = 2.41 min; 2.56 min. (ESI):
m/z = 428 [M + H]+.
Diethyl (3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
phosphonate (38). According to the general coupling procedure 4-
chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.165 g, 0.75 mmol) and
diethyl (3-aminobenzyl)phosphonate·HCl 35 (0.252 g, 0.9 mmol) were
reacted. The crude product was purified by preparative TLC in
EtOAc:EtOH (9:1). The product was extracted from the silica with
MeOH. After evaporation, the residue was dissolved in EtOAc (100
mL), washed with 1 M aqueous HCl (100 mL) with satd aqueous
NaHCO3 (100 mL) with brine and then dried with MgSO4. After
concentration, a yellow powder was obtained (0.041 g, 13%); mp =
217−218 °C (EtOAc). 31P NMR (CDCl3, 121 MHz): δ 29.4 ppm. 1H
NMR (DMSO-d6, 300 MHz): δ 9.62 (s, 1H), 8.68 (s, 1H), 7.94 (dd,
3JHH = 7.7 Hz, 4JHH = 1.5 Hz, 1H), 7.55−7.66 (m, 2H), 7.45 (s, 2H), 7.27
Diethyl [Hydroxy(4-{[6-(2-methoxyphenyl)pyrimidin-4-yl]amino}-
phenyl)methyl]phosphonate (41b). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.221 g, 1.0
mmol) and diethyl [(4-aminophenyl)(hydroxy)methyl]phosphonate·
HCl 34 (0.44 g, 1.5 mmol) were reacted. The crude product was
recrystallized first from hexane/CH2Cl2 then from MeCN to give a
yellow powder (0.140 g, 32%); mp = 195−196 °C (MeCN). 31P NMR
1
(DMSO-d6, 242 MHz): δ 21.0 ppm (82%) and 7.4 ppm (18%). H
NMR (DMSO-d6, 300 MHz): δ 9.62 (s, 1H), 8.69 (s, 1H), 7.95 (d, 3JHH
= 5.2 Hz, 1H), 7.55−7.7 (m, 2H), 7.3−7.5 (m, 4H), 7.18 (d, 3JHH = 6.4
Hz, 1H), 7.08 (s, 1H), 6.09 (d, 3JPH = 12.4 Hz, 1H), 4.88 (d, 2JPH = 11.1
Hz, 1H), 3.9−4.05 (m, 7H), 1.17 (s, 6H). Peaks are broads, some of
them split in 3 to 1 ratio on heating. We hypothesize that α-hydroxy
protons can form intramolecular H-bonds with the oxygen of
phosphorus, and these asymmetric ring formation causes odd spectra.
Derivatives without α-hydroxy group never show this phenomenon. LC-
MS: Rt = 0.46 min; 2.14 min; 2.32 min. (ESI): m/z = 444 [M + H]+.
Diethyl (4-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}benzyl)-
phosphonate (42a). According to the general coupling procedure, 4-
chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.236 g, 1.0 mmol) and
diethyl (4-aminobenzyl)phosphonate·HCl 36 (0.419 g, 1.5 mmol) were
reacted. Purification was different from the general procedure: after the
suspension of the crude product in satd NaHCO3, EtOAc (0.5 mL) was
added and the mixture was stirred at 0 °C for 30 min. The precipitate was
filtered and washed with Et2O. The desired compound was isolated as a
yellow powder (0.44 g, 99%); mp = 204 °C (water, EtOAc). 31P NMR
(DMSO-d6, 242 MHz): δ 25.8 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.81 (s, 1H), 8.81 (s, 1H), 8.75 (s, 1H), 8.43 (d, 3JHH = 7.7 Hz, 1H), 8.35
(d, 3JHH = 8.1 Hz, 1H), 7.83 (t, 3JHH = 7.9 Hz, 1H), 7.65 (d, JAABB = 8.1
Hz, 2H), 7.36 (s, 1H), 7.25 (dd, JAABB = 8.7 Hz, J = 2.1 Hz, 2H), 3.89−
4.00 (m, 4H), 3.18 (d, 2JPH = 21.2 Hz, 2H), 1.17 (t, 3JHH = 7.0 Hz, 6H).
LC-MS: Rt = 3.62 min. (ESI): m/z = 443 [M + H]+.
(t, 3JHH = 7.7 Hz, 0.1H), 7.18 (d, 3JHH = 8.3 Hz, 1H), 7.08 (t, 3JHH = 7.5
Hz, 1H), 6.93 (d, 3JHH = 7.6 Hz, 1H), 3.80−4.00 (m, 4H), 3.90 (s, 3H),
3.20 (d, 2JPH = 22.1 Hz, 2H), 1.18 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt =
2.58 min; 2.73 min. (ESI): m/z = 428 [M + H]+.
Diethyl (3-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}benzyl)-
phosphonate (39a). According to the general coupling procedure, 4-
chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.230 g, 0.98 mmol) and
diethyl (3-aminobenzyl)phosphonate·HCl 35 (0.408 g, 1.46 mmol)
were reacted. The crude product was recrystallized from MeCN and
washed with Et2O to give a yellow powder (0.228 g, 53%); mp = 188−
1
190 °C (MeCN). 31P NMR (DMSO-d6, 242 MHz): δ 25.6 ppm. H
NMR (DMSO-d6, 300 MHz): δ 9.83 (s, 1H), 8.83 (s, 1H), 8.76 (s, 1H),
8.47 (d, 3JHH = 7.6 Hz, 1H), 8.36 (d, 3JHH = 7.9 Hz, 1H), 7.83 (t, 3JHH
=
7.9 Hz, 1H), 7.59−7.70 (m, 2H), 7.40 (s, 1H), 7.29 (t, 3JHH = 7.5 Hz,
1H), 6.97 (d, 3JHH = 6.9 Hz, 1H), 3.92−4.03 (m, 4H), 3.24 (d, 2JPH
=
21.5 Hz, 2H), 1.19 (t, 3JHH = 6.9 Hz, 6H). LC-MS: Rt = 3.66 min. (ESI):
m/z = 443 [M + H]+.
Diethyl [Hydroxy(3-{[6-(3-nitrophenyl)pyrimidin-4-yl]amino}-
phenyl)methyl]phosphonate (39b). According to the general coupling
procedure, 4-chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.230 g, 0.98
mmol) and diethyl [(3-aminophenyl)(hydroxy)methyl]phosphonate·
HCl 33 (0.433 g, 1.46 mmol) were reacted. The crude product was
recrystallized from MeCN and washed with Et2O to give a yellow
powder (0.329 g, 73%); mp = 191 °C (MeCN). 31P NMR (DMSO-d6,
1
242 MHz): δ 20.8 ppm. H NMR (DMSO-d6, 300 MHz): δ 9.85 (s,
Diethyl [Hydroxy(4-{[6-(3-nitrophenyl)pyrimidin-4-yl]amino}-
phenyl)methyl]phosphonate (42b). According to the general coupling
procedure, 4-chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.220 g, 0.93
mmol) and diethyl [(4-aminophenyl)(hydroxy)methyl]phosphonate·
HCl 34 (0.363 g, 1.4 mmol) were reacted. The crude product was
purified by column chromatography in 100% EtOAc. Fractions
containing the desired compound were combined and concentrated.
The product was isolated after recrystallization from MeCN as a pale-
1H), 8.84 (s, 1H), 8.77 (s, 1H), 8.48 (d, 3JHH = 7.8 Hz, 1H), 8.36 (dd,
3JHH = 8.1 Hz, 4JHH = 1.5 Hz, 1H), 7.84 (t, 3JHH = 8.0 Hz, 1H), 7.69−7.78
(m, 2H), 7.41 (s, 1H), 7.33 (t, 3JHH = 8.1 Hz, 1H), 7.12 (d, 3JHH = 7.4 Hz,
1H), 6.21 (dd, 3JPH = 16.2 Hz, 3JHH = 5.5 Hz, 1H), 4.95 (dd, 2JPH = 13.4
Hz, 3JHH = 5.5 Hz, 1H), 3.88−4.06 (m, 4H), 1.18 (t, 3JHH = 6.9 Hz, 6H).
LC-MS: Rt = 3.26 min. (ESI): m/z = 459 [M + H]+.
Diethyl (3-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}benzyl)-
phosphonate (40a). According to the nitro group reduction described
for compound 26a: reduction of compound 39a (0.100 g, 0.23 mmol)
gave a yellow powder (0.075 g, 73%); mp = 186 °C (Et2O). 31P NMR
(DMSO-d6, 242 MHz): δ 28.5 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
11.02 (s, 1H), 8.84 (s, 1H), 7.45−7.70 (m, 5H), 7.30−7.40 (m, 3H),
7.05−7.15 (m, 1H), 3.95−4.00 (m, 4H), 3.26 (d, 2JPH = 21.0 Hz, 2H),
1.19 (s, 6H). LC-MS: Rt = 2.38 min; 2.48 min. (ESI): m/z = 413 [M +
H]+.
1
yellow powder (0.024 g, 6%); mp = 205 °C, dec (MeCN). H NMR
(DMSO-d6, 300 MHz): δ 9.81 (s, 1H), 8.82 (s, 1H), 8.78 (s, 1H), 8.45
(s, 1H), 8.37 (s, 1H), 7.84 (s, 1H), 7.69 (s, 2H), 7.4 (br, 3H), 6.11 (d,
3JPH = 14.7 Hz, 1H), 4.90 (d, 2JPH = 9.8 Hz, 1H), 3.98 (br, 4H), 1.18 (br,
6H). The spectrum has the similar phenomena as those of compound
41b. LC-MS: Rt = 3.22 min. (ESI): m/z = 459 [M + H]+.
Diethyl (4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}benzyl)-
phosphonate (43). According to the nitro group reduction described
for compound 26a: reduction of compound 42a (0.300 g, 0.68 mmol)
afforded a yellow powder (0.182 g, 65%); mp = 62−65 °C (EtOAc),
Diethyl [(3-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl)-
(hydroxy)methyl]phosphonate (40b). According to the nitro group
reduction described for compound 26a: reduction of compound 39b
(0.100 g, 0.22 mmol) afforded a yellow powder (0.082 g, 80%); mp =
265−267 °C (Et2O). 31P NMR (DMSO-d6, 242 MHz): δ 23.6 ppm. 1H
NMR (DMSO-d6, 300 MHz): δ 11.14 (s, 1H), 8.86 (s, 1H), 7.10−7.80
1
amorph. 31P NMR (DMSO-d6, 242 MHz): δ 25.8 ppm. H NMR
(DMSO-d6, 300 MHz): δ 9.57 (s, 1H), 8.64 (s, 1H), 7.62 (d, JAABB = 8.2
Hz, 2H), 7.21−7.27 (m, 3H), 7.09−7.16 (m, 3H), 6.67 (d, 3JHH = 6.8
2
Hz, 1H), 5.26 (br, 2H), 3.88−3.99 (m, 4H), 3.15 (d, JPH = 21.2 Hz,
2
(m, 9H), 6.00 (br, 3H), 4.98 (d, JPH = 13.2 Hz, 1H), 3.95−4.00 (m,
2H), 1.17 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt = 0.46 min; 2.22 min; 2.44
min. (ESI): m/z = 413 [M + H]+.
4H), 1.17 (s, 6H). LC-MS: Rt = 2.01 min; 2.23 min. (ESI): m/z = 429
[M + H]+.
Diethyl (5-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-2-
methylbenzyl)phosphonate (44). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.165 g,
0.75 mmol) and diethyl (5-amino-2-methylbenzyl)phosphonate·HCl
37 (0.072 g, 0.25 mmol) were reacted. The crude product was purified
Diethyl (4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
phosphonate (41a). According to the general coupling procedure, 4-
chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.221 g, 1.0 mmol) and
diethyl (4-aminobenzyl)phosphonate·HCl 36 (0.419 g, 1.5 mmol) were
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by preparative TLC with EtOAc:hexane (8:1). The pure product was
extracted from the silica with MeOH. After evaporation of solvents
under vacuum, a yellow powder was obtained (0.04 g, 53%); mp = 134−
(DMSO-d6, 300 MHz): δ 8.9 (br, 2H), 7.63−7.77 (m, 2H), 7.57 (d, 3JHH
= 5.9 Hz, 1H), 7.40−7.60 (m, 2H), 7.15 (t, 3JHH = 7.0 Hz, 1H), 6.80−
7.00 (m, 2H), 6.83 (d, 3JHH = 4.8 Hz, 1H), 3.75−3.85 and 3.85−3.95 (2
× m, 2 × 1H), 3.42 (d, 2JPH = 17.2 Hz, 2H), 1.16 (t, 3JHH = 6.7 Hz, 3H).
LC-MS: Rt = 0.45 and 2.28 min; (ESI): m/z = 276 [M + H]+.
Ethyl (3-Aminobenzyl)ethylphosphinate (47). Diethyl ethylphos-
phonite was prepared from triethyl-phosphite (16.6 g, 100 mmol) and
ethyl bromide (16.35 g, 150 mmol) according to the method described
by Petnehazy et al.32 The ethylphosphonite was isolated as a colorless oil
(11.25 g, 50%). 31P NMR (CDCl3, 121 MHz): δ 185.6 ppm. 1H NMR
1
137 °C (MeOH). 31P NMR (DMSO-d6, 242 MHz): δ 28.9 ppm. H
NMR (DMSO-d6, 300 MHz): δ 9.56 (s, 1H), 8.56 (s, 1H), 7.93 (dd,
3JHH = 7.7 Hz, 4JHH = 1.5 Hz, 1H), 7.41−7.56 (m, 3H), 7.42 (s, 1H), 7.17
(d, 3JHH = 8.3 Hz, 1H; 7.13 (d, 3JHH = 8.1 Hz, 1H), 7.07 (t, 3JHH = 7.4 Hz,
1H), 3.89−4.00 (m, 4H), 3.89 (s, 3H), 3.17 (d, 2JPH = 21.6 Hz, 2H), 2.29
(s, 3H), 1.18 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt = 2.92 min. (ESI): m/z =
442 [M + H]+.
(CDCl3, 300 MHz): δ 3.72−3.97 (m, JPH = 12.1 Hz, 4H), 1.86 (dt, JHH
=
(4-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}benzyl)phosphonic
Acid (45a). Diethyl (4-{[6-(3-nitrophenyl)pyrimidin-4-yl]amino}-
benzyl)phosphonate 42a (0.100 g, 0.23 mmol) was refluxed in 5 M
aqueous HCl solution (30 mL) for 24 h. Volatiles were evaporated then
the residue was dissolved in water (25 mL) and it was washed with DEE
(3 × 100 mL). Water was evaporated then dry toluene (20 mL) was
distilled from the residue. The dry residue was dissolved in abs EtOH (5
mL) and cooled to −9 °C then propylene-oxide (0.5 mL) was added to
the mixture. After 15 min stirring, the precipitate was filtered and washed
with Et2O. Compound 45a was isolated as a yellow powder (0.059g,
66%); mp = 265−270 °C, dec (EtOH). 31P NMR (DMSO-d6, 242
MHz): δ 20.1 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 10.48 (s, 1H),
8.79 (s, 1H), 8.77 (s, 1H), 8.39 (dd, 3JHH = 3JHH = 7.8 Hz, 2H), 7.85 (t,
3JHH = 8.0 Hz, 1H), 7.63 (d, JAABB = 7.8 Hz, 2H), 7.15 (s, 1H), 7.24 (d,
6.4 Hz, JPH = 12.8 Hz, 2H), 1.26 (t, JHH = 7.1 Hz, 6H), 1.05 (dt, JHH = 6.5
Hz, JPH = 14.3 Hz, 3H). 3-Nitro-benzylbromide (1.08 g; 5.0 mmol) and
diethyl ethylphosphonite (1.125 g; 7.5 mmol) were heated to 140 °C
under argon flow for 1 h. When the reaction mixture was cooled to 50−
60 °C and diluted with CHCl3 (5 mL) then it was cooled further to 0 °C
and 5 M aqueous HCl solution (2 mL) was added. After 15 min of
stirring, the mixture was diluted with water (10 mL) and was extracted
with CHCl3 (3 × 50 mL). Combined organic phases were washed with 2
M aqueous NaOH solution (2 × 50 mL) and with brine then it was dried
with Mg SO4. After evaporation of the volatiles under reduced pressure,
the residue was suspended in MeOH (100 mL) and it was discolored by
charcoal. After filtration on a celite pad and concentration, the
phosphinate was obtained as a yellow oil (0.846 g, 66%). It was used
in the next step without further purification. 31P NMR (DMSO-d6, 242
MHz): δ 54.9 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 8.18 (s, 1H),
J
AABB = 7.5 Hz, 2H), 5.2 (br, 2H), 2.93 (d, 2JPH = 21.4 Hz, 2H). LC-MS:
Rt = 2.40 min. (ESI): m/z = 387 [M + H]+.
8.10 (d, 3JHH = 7.0 Hz, 1H), 7.73 (d, 3JHH = 7.2 Hz, 1H), 7.62 (t, 3JHH
=
(4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}benzyl)phosphonic
Acid (45b). Compound 45b was prepared with the method described
for compound 45a starting from diethyl phosphonate 43 (0.128 g, 0.31
mmol), yielding a yellow powder (0.11 g, 99%); mp> 300 °C (EtOH).
7.5 Hz, 1H), 3.80−4.00 (m, 2H), 3.40 (d, 2JPH = 16.4 Hz, 2H), 1.60−
1.70 (m, 2H), 1.16 (s, 3H), 1.00 (dt, 3JPH = 18.1 Hz, 3JHH = 7.4 Hz, 3H).
LC-MS: Rt = 2.91 min. (ESI): m/z = 258 [M + H]+. Reduction of ethyl
ethyl [(3-nitrobenzyl)phosphinate (0.837 g, 3.26 mmol) to compound
47 was carried out according to the nitro group reduction described for
compound 26a, giving a yellow oil (0.69 g, 93%). 31P NMR (DMSO-d6,
1
31P NMR (DMSO-d6, 242 MHz): δ 20.4 ppm. H NMR (DMSO-d6,
300 MHz): δ 11.08 (s, 1H), 8.73 (s, 1H), 7.52 (d, JAABB = 8.1 Hz, 2H),
7.38−7.48 (m, 3H), 7.24 (d, JAABB = 8.1 Hz, 2H), 7.12−7.20 (m, 1H),
7.14 (s, 1H), 6.30 (br, 4H), 2.98 (d, 2JPH = 21.4 Hz, 2H). LC-MS: Rt =
0.46 min; 1.64 min. (ESI): m/z = 357 [M + H]+.
1
242 MHz): δ 56.1 ppm. H NMR (DMSO-d6, 300 MHz): δ 6.92 (s,
1H), 6.47 (s, 1H), 6.41 (s, 2H), 5.07 (br, 2H), 3.80−3.90 (m, 2H), 2.97
(d, 2JPH = 16.5 Hz, 2H), 1.50−1.60 (m, 2H), 1.18 (s, 3H), 0.97 (dt, 3JPH
= 17.6 Hz, 3JHH ∼ 8.7 Hz, 3H). LC-MS: Rt = 0.45 and 1.73 min. (ESI):
m/z = 228 [M + H]+.
Ethyl (3-Aminobenzyl)phenylphosphinate (46). Ethyl phenyl-
phosphinate (1.02 g, 6.0 mmol) was dissolved in anhydrous THF (20
mL) then triethylamine (0.97 mL; 7.0 mmol) and trimethylsilyl chloride
(0.89 mL; 7.0 mmol) were added to the solution, respectively. The
mixture was stirred for 1 h at RT then the precipitate was filtered off.
Filtrate was evaporated under reduced pressure then anhydrous toluene
(30 mL) was evaporated from the residue. To the obtained yellow oil, 3-
nitrobenzyl bromide (1.08 g; 5.0 mmol) was added and the mixture was
heated to 140 °C under argon flow for 4.5 h. When the reaction mixture
was cooled to 50−60 °C and diluted with CHCl3 (5 mL) then it was
cooled further to 0 °C and 5 M aqueous HCl solution (2 mL) was added.
After 15 min of stirring, the mixture was diluted with water (10 mL) and
was extracted with CHCl3 (3 × 50 mL). Combined organic phases were
washed with 2 M aqueous NaOH solution (2 × 50 mL) and with brine
then it was dried with Mg SO4. After evaporation of the solvent, the
phosphinic acid was obtained as a yellow powder (0.645 g, 47%); mp =
127−128 °C (CHCl3). 31P NMR (DMSO-d6, 242 MHz): δ 54.9 ppm.
1H NMR (DMSO-d6, 300 MHz): δ 8.03 (d, 3JHH = 5.4 Hz, 1H), 7.98 (s,
1H), 7.45−7.75 (m, 7H), 3.43 (d, 2JPH = 17.3 Hz, 2H). LC-MS: Rt = 2.62
min. (ESI): m/z = 278 [M + H]+. (3-Nitrobenzyl)phenylphosphinic
acid (0.880 g; 3.18 mmol) was dissolved in DMF (10 mL) then triethyl
orthoformate (5.28 mL; 31.80 mmol) and one drop trifluoroacetic acid
was added. The reaction mixture was stirred for 1 week at 80 °C, and
then it was evaporated. The residue was suspended in 2 M aqueous
NaOH solution (50 mL), and it was extracted with EtOAc (3 × 75 mL).
The combined organic layers were washed with brine and dried with
MgSO4. After evaporation, the phosphonic ester was obtained as a
yellow oil (0.729 g, 75%). 1H NMR (DMSO-d6, 300 MHz): δ 8.03 (d,
3JHH = 5.1 Hz, 1H), 8.00 (s, 1H), 7.60−7.75 (m, 2H), 7.45−7.60 (m,
5H), 3.80−3.97 and 3.60−3.75 (2xm, 2 × 1H), 3.66 (d, 2JPH = 17.3 Hz,
2H), 1.18 (t, 3JHH = 6.9 Hz, 3H). LC-MS: Rt = 3.45 min. (ESI): m/z =
306 [M + H]+. Reduction of ethyl (3-nitrobenzyl)phenylphosphinate
(0.72 g, 2.36 mmol) to compound 46 was carried out according to the
nitro group reduction described for compound 26a giving a yellow oil
(0.586 g, 90%). 31P NMR (242 MHz): DMSO-d6, δ 41.5 ppm. 1H NMR
Ethyl (3-Aminobenzyl)propylphosphinate (48). The diethyl
propylphosphonite was prepared from (18.45 g; 150 mmol) propyl
bromide and (16.6 g; 100 mmol) triethyl-phosphite according to the
method described by Petnehazy et al.32 The propylphosphonite was
isolated as a colorless oil (17.71 g, 72%). 31P NMR (CDCl3, 121 MHz):
δ 184.9 ppm. 1H NMR (CDCl3, 300 MHz): δ 3.70−3.91 (m, 4H), 1.85
(dt, JHH = 6.6 Hz, JPH = 12.0 Hz, 2H), 1.42−1.51 (m, 2H), 1.24 (t, JHH
=
7.0 Hz, 6H), 0.99 (t, JHH = 6.6 Hz, 3H). Diethyl propylphosphonite
(2.61 g, 17.4 mmol) was reacted with 3-nitrobenzyl bromide (2.51 g,
11.6 mmol) as it is described for compound 47. The propylphosphinate
was isolated as a brown oil (2.88 g; 92%) and was used without further
1
purification. 31P NMR (CDCl3, 121 MHz): δ 50.8 ppm. H NMR
(CDCl3, 300 MHz): δ 8.17 (d, 4JHH = 1.2 Hz, 1H), 8.11 (d, 3JHH = 6.9
Hz, 1H), 7.70 (d, 3JHH = 7.5 Hz, 1H), 7.54 (t, 3JHH = 7.2 Hz, 1H), 3.94−
4.16 (m, 2H), 3.27 (d, 2JPH = 15.6 Hz, 2H), 1.56−1.80 (m, 4H), 1.27 (t,
3JHH = 6.9 Hz, 3H), 1.02 (t, 3JHH = 7.2 Hz, 3H). Reduction of ethyl (3-
nitrobenzyl)propylphosphinate (0.88 g, 10.6 mmol) to compound 48
was carried out according to the nitro group reduction described for
compound 26a, yielding a yellow oil (2.148 g, 84%). 31P NMR (CDCl3,
121 MHz): δ 53.1 ppm. 1H NMR (CDCl3, 300 MHz): δ 7.07 (t, 3JHH
=
7.8 Hz, 1H), 6.68 (d, 4JHH = 1.5 Hz, 1H), 6.63 (d, 3JHH = 8.4 Hz, 1H),
6.60 (d, 3JHH = 8.4 Hz, 1H), 3.95−4.05 (m, 2H), 3.03 (d, 2JPH = 16.8 Hz,
2H), 1.50−1.70 (m, 4H), 1.26 (t, 3JHH= 7.2 Hz, 3H), 0.96 (t, 3JHH = 6.9
Hz, 3H).
Ethyl (4-Aminobenzyl)ethylphosphinate (49). The 4-nitrobenzyl
bromide (1.296 g 6.0 mmol) and 1.35 g diethyl ethylphosphonite (9.0
mmol) were reacted according to the preparation of 47, giving a yellow
oil (0.847 g, 55%). 31P NMR (DMSO-d6, 242 MHz): δ 54.7 ppm. 1H
NMR (DMSO-d6, 300 MHz): δ 8.18 (d, JAABB = 7.6 Hz, 2H), 7.55 (d,
JAABB = 6.6 Hz, 2H), 3.90−3.93 (m, 2H), 3.39 (d, 2JPH = 16.8 Hz, 2H),
1.60−1.68 (m, 2H), 1.16 (t, 3JHH = 6.8 Hz, 3H), 1.00 (dt, 3JPH = 18.1 Hz,
3JHH = 7.4 Hz, 3H). LC-MS: Rt = 2.88 min. (ESI): m/z = 258 [M + H]+.
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Journal of Medicinal Chemistry
Article
Reduction of ethyl ethyl (4-nitrobenzyl)phosphinate (0.843 g, 3.28
mmol) was carried out according to the nitro group reduction described
for compound 26a, providing a brown oil (0.375 g, 50%). LC-MS: Rt =
0.44 min. (ESI): m/z = 228 [M + H]+. Compound 49 was used without
further purification and characterization.
Ethyl (3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
phenylphosphinate (52c). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.157 g,
0.71 mmol) and ethyl (3-aminobenzyl)phenylphosphinate·HCl 46
(0.332 g, 1.07 mmol) were reacted. The crude product was purified by
preparative TLC with EtOAc:hexane (8:1) and 1 v/v% AcOH as eluent.
The product was extracted with MeOH from the silica. After
evaporation, the residue was dissolved in EtOAc (100 mL) and washed
with 0.1 M aqueous AcOH solution (3 × 30 mL), with satd aqueous
NaHCO3 solution (100 mL), and with brine (100 mL). The organic
phase was dried with MgSO4 and evaporated under reduced pressure.
52c was obtained as yellow powder (0.094 g, 29%); mp = 103−104 °C
Ethyl (4-Aminobenzyl)propylphosphinate (50). The 4-nitro-benzyl
bromide (1.296 g, 6.0 mmol) and diethyl propylphosphonite (1.476 g;
9.0 mmol) were reacted according to the preparation of 47, yielding a
brown oil (1.426 g; 88%). 31P NMR (DMSO-d6, 242 MHz): δ 53.1 ppm.
1H NMR (DMSO-d6, 300 MHz): δ 8.18 (d, JAABB = 7.6 Hz, 2H), 7.55 (d,
J
AABB = 7.5 Hz, 2H), 3.90−3.95 (m, 2H), 3.39 (d, 2JPH = 17.0 Hz, 2H),
1.55−1.65 (m, 2H), 1.40−1.50 (m, 2H), 1.13−1.22 (m, 3H), 0.93 (br,
3H). LC-MS: Rt = 3.16 min. (ESI): m/z = 272 [M + H]+. Reduction of
ethyl (4-nitrobenzyl)propylphosphinate (1.42 g, 5.24 mmol) was
carried out according to the nitro group reduction described for
compound 26a, giving a brown oil (1.005 g; 80%). LC-MS: Rt = 0.44 and
1.89 min. (ESI): m/z = 242 [M − H]+. Compound 50 was used without
further purification and characterization.
1
(EtOAc). 31P NMR (DMSO-d6, 242 MHz): δ 41.5 ppm. H NMR
(DMSO-d6, 300 MHz): δ 9.56 (s, 1H), 8.64 (s, 1H), 7.93 (d, 3JHH = 6.2
Hz, 1H), 7.30−7.75 (m, 8H), 7.00−7.20 (m, 4H), 6.73 (d, 3JHH = 3.9
Hz, 1H), 3.90−4.00 (m, 1H), 3.90 (s, 3H), 3.80−3.88 (m, 1H), 3.42 (d,
2JPH = 17.3 Hz, 2H), 1.18 (t, 3JHH = 6.0 Hz, 3H). LC-MS: Rt = 2.92 min.
(ESI): m/z = 460 [M + H]+.
Ethyl Ethyl (3-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}benzyl)-
phosphinate (53a). According to the general coupling procedure, 4-
chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.118 g, 0.5 mmol) and ethyl
(3-aminobenzyl)ethylphosphinate·HCl 47 (0.158 g, 0.6 mmol) were
reacted. The crude product was recrystallized from MeCN to give a
yellow powder (0.118 g, 55%); mp = 189−190 °C (MeCN). 31P NMR
(DMSO-d6, 242 MHz): δ 55.8 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.81 (s, 1H), 8.84 (s, 1H), 8.76 (s, 1H), 8.48 (d, 3JHH = 6.9 Hz, 1H), 8.36
(d, 3JHH = 7.5 Hz, 1H), 7.84 (t, 3JHH = 7.7 Hz, 1H), 7.55−7.65 (m, 2H),
7.41 (s, 1H), 7.30 (t, 3JHH = 7.2 Hz, 1H), 6.99 (d, 3JHH = 4.8 Hz, 1H),
3.90−4.00 (m, 2H), 3.19 (d, 2JPH = 16.5 Hz, 2H), 1.60−1.70 (m, 2H),
1.19 (t, 3JHH = 6.3 Hz, 3H), 1.01 (dt, 3JPH = 17.7 Hz, 3JHH = 8.8 Hz, 3H).
LC-MS: Rt = 3.41 min. (ESI): m/z = 427 [M + H]+.
Ethyl (5-Amino-2-methylbenzyl)ethylphosphinate (51). The 2-
(bromomethyl)-1-methyl-4-nitrobenzene (1.150 g, 5.0 mmol, issued
from the synthesis of 37) and diethyl ethylphosphonite (1.294 g; 7.5
mmol) were reacted according to the reaction described for compound
1
47, affording a brown oil (0.918 g, 68%). H NMR (DMSO-d6, 300
MHz): δ 7.78 (s, 1H), 7.66 (d, 3JHH = 6.9 Hz, 1H), 7.38 (d, 3JHH = 7.2
Hz, 1H), 3.80−4.10 (m, 2H), 3.30 (2H, overlaps with DMSO’s water),
2.44 (s, 3H), 1.65−1.70 (m, 2H), 1.13 (t, 3JHH = 6.9 Hz, 3H), 1.04 (dt,
3JPH = 17.7 Hz, 3JHH = 8.1 Hz, 3H). Reduction of ethyl ethyl (2-methyl-
5-nitrobenzyl)phosphinate (0.90 g, 3.32 mmol) was carried out
according to the nitro group reduction described for compound 26a,
giving a brown oil (0.411 g, 51%). 1H NMR (DMSO-d6, 300 MHz): δ
6.78 (d, 3JHH = 7.1 Hz, 1H), 6.45 (s, 1H), 6.34 (d, 3JHH = 5.8 Hz, 1H),
4.70 (br, 2H), 3.80−4.00 (m, 2H), 2.97 (d, 2JPH = 16.7 Hz, 2H), 1.91 (s,
Ethyl (3-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (53b). According to the general coupling
procedure, 4-chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.22 g, 0.92
mmol) and ethyl (3-aminobenzyl)propylphosphinate·HCl 48 (0.25 g,
1.04 mmol) were reacted. After the reaction the crude product was
filtered from the NaHCO3 suspension. The resulting solid was
recrystallized from MeCN to give a yellow powder (0.12 g, 29%); mp
= 173−176 °C (MeCN). 31P NMR (DMSO-d6, 242 MHz): δ 54.3 ppm.
1H NMR (DMSO-d6, 300 MHz): δ 9.82 (s, 1H), 8.84 (s, 1H), 8.76 (s,
3
3H), 1.55−1.75 (m, 2H), 1.16 (br, 3H), 1.00 (t, JHH = 7.5 Hz, 3H).
1
According to the H NMR spectra purity of compound 51 was about
50%. It was used without further purification in the next step. LC-MS: Rt
= 0.44 min. (ESI): m/z = 242 [M + H]+.
Ethyl Ethyl (3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-
benzyl)phosphinate (52a). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.075 g,
0.34 mmol) and ethyl (3-aminobenzyl)ethylphosphinate·HCl 47 (0.108
g, 0.41 mmol) were reacted. The crude product was purified by
preparative TLC using EtOAc:hexane (8:1) and 1 v/v% AcOH as
eluent. The product was extracted from the silica with MeOH. After
concentration, 52a was dissolved in EtOAc (100 mL) and it was washed
with 0.1 M aqueous AcOH solution (3 × 30 mL), with satd aqueous
NaHCO3 solution (100 mL), and with brine (100 mL). The organic
phase was dried with MgSO4 and evaporated under vacuum. 52a was
obtained as yellow powder (0.045 g, 32%); mp = 30 °C. 31P NMR
(DMSO-d6, 242 MHz): δ 55.6 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.62 (s, 1H), 8.67 (s, 1H), 7.94 (d, 3JHH = 6.2 Hz, 1H), 7.55−7.68 (m,
2H), 7.45 (s, 2H), 7.27 (t, 3JHH = 7.7 Hz, 1H), 7.17 (d, 3JHH = 7.5 Hz,
1H), 7.08 (t, 3JHH = 7.5 Hz, 1H), 6.93 (d, 3JHH = 7.6 Hz, 1H), 3.80−4.00
(m, 2H), 3.90 (s, 3H), 3.17 (d, 2JPH = 16.5 Hz, 2H), 1.50−1.70 (m, 2H),
1.18 (t, 3JHH = 7.0 Hz, 3H), 1.00 (dt, 3JPH = 17.6 Hz, 3JHH = 7.2 Hz, 3H).
LC-MS: Rt = 2.56 min. (ESI): m/z = 412 [M + H]+.
1H), 8.48 (d, 3JHH = 6.8 Hz, 1H), 8.36 (d, 3JHH = 6.2 Hz, 1H), 7.83 (t,
3JHH = 7.7 Hz, 1H), 7.55−7.65 (m, 2H), 7.42 (s, 1H), 7.30 (s, 1H), 6.99
(s, 1H), 3.90−4.00 (m, 2H), 3.18 (d, 2JPH = 16.7 Hz, 2H), 1.50−1.70 (m,
4H), 1.19 (s, 3H), 0.93 (s, 1H). LC-MS: Rt = 3.63 min. (ESI): m/z = 441
[M + H]+.
Ethyl (3-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}benzyl)-
phenylphosphinate (53c). According to the general coupling
procedure, 4-chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.118 g, 0.5
mmol) and ethyl (3-aminobenzyl)phenylphosphinate·HCl 46 (0.187 g,
0.6 mmol) were reacted. The crude product was recrystallized from
MeCN to give a yellow powder (0.101 g, 43%); mp = 188−190 °C
1
(MeCN). 31P NMR (DMSO-d6, 242 MHz): δ 41.7 ppm. H NMR
(DMSO-d6, 300 MHz): δ 9.76 (s, 1H), 8.84 (s, 1H), 8.73 (s, 1H), 8.48
(d, 3JHH = 6.3 Hz, 1H), 8.36 (d, 3JHH = 7.1 Hz, 1H), 7.84 (t, 3JHH = 6.6
Hz, 1H), 7.65−7.75 (m, 2H), 7.40−7.60 (m, 5H), 7.39 (s, 1H), 7.18 (t,
3
3JHH = 6.9 Hz, 1H), 6.79 (d, JHH = 5.1 Hz, 1H), 3.90−3.95 (m, 1H),
Ethyl (3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (52b). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.17 g;
0.77 mmol) and ethyl (3-aminobenzyl)propylphosphinate·HCl 48
(0.229 g; 0.83 mmol) were reacted. The crude product was purified by
column chromatography with 100% EtOAc to EtOAc:EtOH (1:1).
After concentration, 52b was obtained as a yellow oil (0.16 g, 50%). 31P
3.80−3.85 (m, 1H), 3.45 (d, 2JPH = 17.3 Hz, 2H), 1.18 (s, 3H). LC-MS:
Rt = 3.83 min. (ESI): m/z = 475 [M + H]+.
Ethyl (3-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}benzyl)-
ethylphosphinate (54a). Reduction of nitro compound 53a (0.099 g,
0.23 mmol) was carried out according to the nitro group reduction
described for compound 26a, yielding a yellow powder (0.079 g, 79%);
mp = 208−211 °C (EtOH, Et2O). 31P NMR (DMSO-d6, 242 MHz): δ
1
NMR (CDCl3, 121 MHz): δ 52.5 ppm. H NMR (DMSO-d6, 300
1
MHz): δ 9.67 (s, 1H), 8.68 (s, 1H), 7.94 (d, 3JHH = 6.2 Hz, 1H), 7.55−
55.6 ppm. H NMR (DMSO-d6, 300 MHz): δ 11.09 (s, 1H), 8.84 (s,
7.68 (m, 2H), 7.45 (s, 2H), 7.28 (t, 3JHH = 7.1 Hz, 1H), 7.17 (d, 3JHH
=
1H), 7.50−7.80 (m, 5H), 7.30−7.45 (m, 3H), 7.09 (d, 3JHH = 5.4 Hz,
1H), 5.30 (br, 2H), 3.80−3.95 (m, 2H), 3.22 (d, 2JPH = 16.2 Hz, 2H),
1.60−1.67 (m, 2H), 1.18 (t, 3JHH = 6.7 Hz, 3H), 1.00 (dt, 3JPH = 17.6 Hz,
3JHH = 7.2 Hz, 3H). LC-MS: Rt = 0.45 and 2.06 and 2.35 min. (ESI): m/z
= 397 [M + H]+.
8.0 Hz, 1H), 7.09 (t, 3JHH = 6.8 Hz, 1H), 6.93 (d, 3JHH = 5.8 Hz, 1H),
3.89 (s, 5H), 3.16 (d, 2JPH = 16.5 Hz, 2H), 1.35−1.70 (m, 4H), 1.18 (t,
3JHH = 6.8 Hz, 3H), 0.92 (br, 3H). LC-MS: Rt = 2.75 min. (ESI): m/z =
426 [M + H]+.
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Journal of Medicinal Chemistry
Article
Ethyl (3-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (54b). Reduction of nitro compound 53b (0.10
g, 0.23 mmol) was carried out according to the nitro group reduction
described for compound 26a, affording a yellow powder (0.079 g, 77%);
mp = 206−208 °C (EtOH, Et2O). 1H NMR (DMSO-d6, 300 MHz): δ
11.02 (s, 1H), 8.83 (s, 1H), 7.65−7.75 (m, 2H), 7.50−7.65 (m, 3H),
7.30−7.45 (m, 3H), 7.05−7.15 (m, 1H). 5.47 (br, 2H), 3.85−4.00 (m,
2H), 3.21 (d, 2JPH = 15.9 Hz, 2H), 1.60−1.70 (m, 2H), 1.45−1.55 (m,
2H), 1.17 (t, 3JHH = 6.9 Hz, 3H), 0.93 (t, 3JHH = 6.3 Hz, 3H). LC-MS: Rt
= 0.43 and 2.37 and 2.55 min. (ESI): m/z = 411 [M + H]+.
MeCN to give a yellow powder (0.085 g, 39%); mp = 219−220 °C
1
(MeCN). 31P NMR (DMSO-d6, 242 MHz): δ 54.3 ppm. H NMR
(DMSO-d6, 300 MHz): δ 9.80 (s, 1H), 8.82 (s, 1H), 8.76 (s, 1H), 8.44
(d, 3JHH = 6.4 Hz, 1H), 8.36 (d, 3JHH = 6.8 Hz, 1H), 7.84 (t, 3JHH = 7.1
Hz, 1H), 7.66 (d, JAABB = 7.0 Hz, 2H), 7.35 (s, 1H), 7.25 (d, JAABB = 5.7
Hz, 2H), 3.90−3.95 (m, 2H), 3.13 (d, 2JPH = 16.2 Hz, 2H), 1.45−1.63
(m, 4H), 1.18 (br, 3H), 0.99 (br, 3H). LC-MS: Rt = 3.58 min. (ESI): m/
z = 441 [M + H]+.
Ethyl (4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}benzyl)-
ethylphosphinate (57a). Reduction of nitro compound 56a (0.081 g;
0.19 mmol) was carried out according to the nitro group reduction
described for compound 26a, giving a yellow powder (0.062 g, 75%);
mp = 152−155 °C (EtOH, Et2O). 31P NMR (DMSO-d6, 242 MHz): δ
Ethyl (3-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}benzyl)-
phenylphosphinate (54c). Reduction of nitro compound 53c (0.08 g,
0.17 mmol) was carried out according to the nitro group reduction
described for compound 26a, giving a yellow powder (0.068 g, 84%);
mp = 208−209 °C (EtOH, Et2O). 31P NMR (DMSO-d6, 242 MHz): δ
1
55.3 ppm. H NMR (DMSO-d6, 300 MHz): δ 11.09 (s, 1H), 8.82 (s,
1H), 7.50−7.70 (m, 5H), 7.25−7.50 (m, 4H), 4.50 (br, 2H), 3.90−3.95
(m, 2H), 3.17 (d, 2JPH = 15.6 Hz, 2H), 1.55−1.65 (m, 2H), 1.18 (t, 3JHH
= 7.0 Hz, 3H), 0.99 (dt, 3JPH = 17.4 Hz, 3JHH = 8.7 Hz, 3H). LC-MS: Rt =
0.44 min, 2.09 and 2.29 min. (ESI): m/z = 397 [M + H]+.
Ethyl (4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (57b). Reduction of nitro compound 56b (0.063
g, 0.14 mmol) was carried out according to the nitro group reduction
described for compound 26a, affording a brown powder (0.053 g, 85%);
mp = 200 °C (EtOH, Et2O). 31P NMR (DMSO-d6, 242 MHz): δ 54.0
ppm. 1H NMR (DMSO-d6, 300 MHz): δ 11.20 (s, 1H), 8.85 (s, 1H),
7.50−7.70 (m, 5H), 7.25−7.50 (m, 4H), 4.50 (br, 2H), 3.80−4.10 (m,
2H), 3.17 (d, 2JPH = 16.2 Hz, 2H), 1.55−1.60 (m, 2H), 1.40−1.50 (m,
2H), 1.18 (t, 3JHH = 7.0 Hz, 3H), 0.92 (t, 3JHH = 7.0 Hz, 3H). LC-MS: Rt
= 0.42 min, 2.36 and 2.51 min. (ESI): m/z = 411 [M + H]+.
Ethyl Ethyl (5-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-2-
methylbenzyl)phosphinate (58). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.117 g, 0.5
mmol) and ethyl (5-amino-2-methylbenzyl)ethylphosphinate·HCl 51
(0.296 g, 1.07 mmol) were reacted. The crude product was purified by
preparative TLC with EtOAc:hexane (8:1) as eluent. The pure product
was extracted from the silica with MeOH. After evaporation, a yellow
powder was obtained (0.053 g, 24%); mp = 94 °C, (MeOH). 31P NMR
(DMSO-d6, 242 MHz): δ 55.6 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.53 (s, 1H), 8.64 (s, 1H), 7.92 (d, 3JHH = 6.8 Hz, 1H), 7.40−7.60 (m,
4H), 7.00−7.20 (m, 3H), 3.85−3.90 (m, 5H), 3.13 (d, 2JPH = 16.3 Hz,
2H), 2.30 (s, 3H), 1.60−1.80 (m, 2H), 1.15 (t, 3JHH = 6.3 Hz, 3H), 1.03
(dt, 3JPH = 17.0 Hz, 3JHH = 8.5 Hz, 3H). LC-MS: Rt = 2.78 min. (ESI): m/
z = 426 [M + H]+.
1
41.7 ppm. H NMR (DMSO-d6, 300 MHz): δ 10.82 (s, 1H), 8.80 (s,
1H), 7.45−7.75 (m, 10H), 7.20−7.45 (m, 3H), 6.89 (d, 3JHH = 6.0 Hz,
1H). 4.25 (br, 2H), 3.90−3.95 (m, 1H), 3.80−3.85 (m, 1H), 3.48 (d,
2JPH = 17.3 Hz, 2H), 1.18 (t, 3JHH = 5.4 Hz, 3H). LC-MS: Rt = 2.73 min.
(ESI): m/z = 445 [M + H]+.
Ethyl Ethyl (4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-
benzyl)phosphinate (55a). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.11 g, 0.5
mmol) and ethyl (4-aminobenzyl)ethylphosphinate·HCl 49 (0.158 g,
0.6 mmol) were reacted. The crude product was purified by preparative
TLC, for the first elution 100% EtOAc, for the second one
EtOAc:hexane (8:1), were used as eluents. The pure product was
extracted with MeOH from the silica. After evaporation, an amorphous
solid was obtained (0.028 g, 14%); mp = 30 °C (MeOH), 31P NMR
(DMSO-d6, 242 MHz): δ 55.6 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.61 (s, 1H), 8.67 (s, 1H), 7.94 (d, 3JHH = 7.1 Hz, 1H), 7.63 (d, JAABB
=
7.3 Hz, 2H), 7.40−7.50 (m, 2H), 7.23 (d, JAABB = 6.6 Hz, 2H), 7.19 (t,
3JHH = 8.1 Hz, 1H), 7.07 (d, 3JHH = 6.6 Hz, 1H), 3.85−4.00 (m, 5H), 3.13
(d, 2JPH = 16.6 Hz, 2H), 1.50−1.65 (m, 2H), 1.18 (t, 3JHH = 6.6 Hz, 3H),
0.98 (dt, 3JPH = 17.4 Hz, 3JHH = 8.4 Hz, 3H). LC-MS: Rt = 2.5 min. (ESI):
m/z = 412 [M + H]+.
Ethyl (4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (55b). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.11 g, 0.5
mmol) and ethyl (4-aminobenzyl)propylphosphinate·HCl 50 (0.167 g;
0.6 mmol) were reacted. The crude product was purified by preparative
TLC, for the first elution 100% EtOAc, for the second one
EtOAc:hexane (8:1), were used as eluents. The pure product was
extracted with MeOH from the silica. After evaporation amorphous
solid was obtained (0.026 g, 12%); mp = 30 °C (MeOH). 31P NMR
(DMSO-d6, 242 MHz): δ 54.1 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
Ethyl Ethyl (2-Methyl-5-{[6-(3-nitrophenyl)pyrimidin-4-yl]amino}-
benzyl)phosphinate (59). According to the general coupling procedure,
4-chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.118 g, 0.5 mmol) and
ethyl (5-amino-2-methylbenzyl)ethylphosphinate·HCl 51 (0.208 g,
0.75 mmol) were reacted. The crude product was recrystallized from
MeCN to give a yellow powder (0.033 g, 15%); mp = 200−203 °C
(MeCN). 1H NMR (DMSO-d6, 300 MHz): δ 9.72 (s, 1H), 8.84 (s, 1H),
8.72 (s, 1H), 8.49 (d, 3JHH = 6.8 Hz, 1H), 8.35 (d, 3JHH = 6.7 Hz, 1H),
7.83 (t, 3JHH = 8.1 Hz, 1H), 7.52 (s, 1H), 7.47 (d, 3JHH = 6.9 Hz, 1H),
7.38 (s, 1H), 7.15 (d, 3JHH = 6.9 Hz, 1H), 3.80−4.00 (m, 2H), 3.17 (d,
2JPH = 15.7 Hz, 2H), 2.31 (s, 3H), 1.60−1.80 (m, 2H), 1.15 (t, 3JHH = 6.3
9.61 (s, 1H), 8.67 (s, 1H), 7.94 (d, 3JHH = 6.2 Hz, 1H), 7.63 (d, JAABB
=
7.2 Hz, 2H), 7.40−7.50 (m, 2H), 7.22 (d, JAABB = 6.8 Hz, 2H), 7.15−
7.20 (m, 1H), 7.07 (t, 3JHH = 6.6 Hz, 1H), 3.85−4.00 (m, 5H), 3.12 (d,
2JPH = 16.3 Hz, 2H), 1.35−1.70 (m, 4H), 1.18 (t, 3JHH = 6.6 Hz, 3H),
0.92 (br, 3H). LC-MS: Rt = 2.48 and 2.68 min. (ESI): m/z = 426 [M +
H]+.
Ethyl Ethyl (4-{[6-(3-Nitrophenyl)prymidin-4-yl]amino}benzyl)-
phosphinate (56a). According to the general coupling procedure, 4-
chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.118 g; 0.5 mmol) and ethyl
(4-aminobenzyl)ethylphosphinate·HCl 49 (0.158 g; 0.6 mmol) were
reacted. The crude product was recrystallized from MeCN to give a
yellow powder (0.104 g, 49%); mp = 226 °C (MeCN). 31P NMR
(DMSO-d6, 242 MHz): δ 55.6 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.8 (s, 1H), 8.82 (s, 1H), 8.76 (s, 1H), 8.44 (d, 3JHH = 6.8 Hz, 1H), 8.36
(d, 3JHH = 6.3 Hz, 1H), 7.84 (t, 3JHH = 6.3 Hz, 1H), 7.65 (d, JAABB = 6.2
Hz, 2H), 7.35 (s, 1H), 7.25 (d, JAABB = 5.4 Hz, 2H), 3.90−3.95 (m, 2H),
3.14 (d, 2JPH = 16.4 Hz, 2H), 1.50−1.70 (m, 2H), 1.18 (br, 3H), 0.99 (dt,
3JPH = 17.4 Hz, 3JHH = 8.7 Hz, 3H). LC-MS: Rt = 3.33 min. (ESI): m/z =
Hz, 3H), 1.05 (dt, 3JPH = 17.9 Hz, 3JHH = 8.9 Hz, 3H). LC-MS: Rt = 3.04
min. (ESI): m/z = 441 [M + H]+.
Ethyl (5-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}-2-
methylbenzyl)ethylphosphynate (60). Reduction of nitro compound
59 (0.023 g, 0.05 mmol) was carried out according to the nitro group
reduction described for compound 26a. The free base was prepared as a
yellow powder (0.018 g, 81%); mp = 180−181 °C (EtOH). 31P NMR
(DMSO-d6, 242 MHz): δ 55.7 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
10.93 (s, 1H), 8.81 (s, 1H), 7.40−7.70 (m, 5H), 7.10−7.40 (m, 3H),
4.20 (br, 2H), 3.80−4.00 (m, 2H), 3.19 (d, 2JPH = 14.7 Hz, 2H), 2.34 (s,
3H), 1.60−1.80 (m, 2H), 1.15 (t, 3JHH = 6.3 Hz, 3H), 1.04 (dt, 3JPH
=
17.7 Hz, 3JHH = 9.0 Hz, 3H). LC-MS: Rt = 0.53 and 1.18 min. (ESI): m/z
427 [M + H]+.
Ethyl (4-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (56b). According to the general coupling
procedure, 4-chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.118 g, 0.5
mmol) and ethyl (4-aminobenzyl)propylphosphinate·HCl 50 (0.167 g,
0.6 mmol) were reacted. The crude product was recrystallized from
= 411 [M + H]+.
(3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinic Acid (61). Chloro (trimethyl)silane (0.076 mL; 0.6
mmol) and potassium iodide (0.02 g; 0.12 mmol) were dissolved in
chloroform (20 mL). The mixture was stirred for 30 min at RT then the
3958
dx.doi.org/10.1021/jm401742r | J. Med. Chem. 2014, 57, 3939−3965

Journal of Medicinal Chemistry
Article
solution of phosphinate 52b (0.05 g, 0.12 mmol in 3 mL CHCl3) was
added. The reaction mixture was refluxed for 30 h, and then additional
chloro (trimethyl)silane (0.076 mL, 0.6 mmol) and potassium iodide
(0.04 g; 0.24 mmol) were added. The reaction mixture was refluxed for
further 24 h then satd NH4Cl (10 mL) was added. After 5 min stirring,
volatiles were evaporated under reduced pressure. The crude product
was suspended in water (20 mL) and was extracted with CHCl3 (3 × 30
mL). The combined organic phases were washed with brine and dried
with MgSO4. After evaporation, the product was further purified by
preparative TLC with 100% EtOH as eluent. The pure product was
extracted from the silica with MeOH. After evaporation under vacuum, a
white powder was obtained (0.017 g, 36%); mp = 122−125 °C
(DMSO-d6, 300 MHz): δ 8.36 (d, JAABB = 6.7 Hz, 2H), 8.02 (dd, 3JPH
∼
JAABB = 9.0 Hz, 2H), 3.92−4.0 (m, 1H), 3.8−3.9 (m, 1H), 1.93−2.05 (m,
2H), 1.21 (t, 3JHH = 6.8 Hz, 3H), 0.95 (dt, 3JPH = 19.2 Hz, 3JHH = 8.0 Hz,
3H). LC-MS: Rt = 2.86 min. (ESI): m/z = 244 [M − H]+. Reduction of
ethyl ethyl (4-nitrophenyl)phosphinate (1.302 g, 5.36 mmol) was
carried out according to the nitro group reduction described for
compound 26a. The free base was prepared as a yellow oil (0.654 g,
49%). 31P NMR (DMSO-d6, 242 MHz): δ 48.8 ppm. 1H NMR (DMSO-
d6, 300 MHz): δ 7.32 (dd, 3JPH∼JAABB = 8.4 Hz, 2H), 6.61 (d, JAABB = 5.7
Hz, 2H), 5.73 (br, 2H), 3.75−3.85 (m, 1H), 3.65−3.75 (m, 1H), 1.72−
1.75 (m, 2H), 1.16 (br, 3H), 0.91 (dt, 3JPH = 18.3 Hz, 3JHH = 8.4 Hz, 3H).
LC-MS: Rt = 2.16 min. (ESI): m/z = 214 [M + H]+.
1
(MeOH). 31P NMR (DMSO-d6, 121 MHz): δ 36.5 ppm. H NMR
Ethyl (4-Aminophenyl)propylphosphinate (64b). According to the
general Pd-catalyzed coupling procedure, 4-bromo-nitrobenzene (2.02
g, 10.0 mmol) and ethyl propylphosphinate 62b (1.496 g, 11.0 mmol)
were reacted, giving a yellow oil (1.305 g, 54%). 1H NMR (DMSO-d6,
300 MHz): δ 8.35 (d, JAABB = 6.8 Hz, 2H), 8.02 (dd, 3JPH ∼ JAABB = 9.0
Hz, 2H), 3.97 (q, 3JHH = 6.9 Hz, 1H), 3.81 (q, 3JHH = 7.0 Hz, 1H), 1.95−
2.05 (m, 2H), 1.30−1.50 (m, 2H), 1.20 (t, 3JHH = 6.8 Hz, 3H), 0.92 (t,
3JHH = 6.5 Hz, 3H). LC-MS: Rt = 3.22 min. (ESI): m/z = 258 [M + H]+.
Reduction of ethyl (4-nitrophenyl)propylphosphinate (1.30 g, 5.06
mmol) was carried out according to the nitro group reduction described
for compound 26a. The free base was prepared as a yellow oil (0.985 g,
86%). 1H NMR (DMSO-d6, 300 MHz): δ 7.32 (dd, 3JPH ∼ JAABB = 9.0
Hz, 2H), 6.60 (d, JAABB = 5.7 Hz, 2H), 5.70 (br, 2H), 3.80−3.85 (m, 1H),
3.60−3.75 (m, 1H), 1.70−1.80 (m, 2H), 1.25−1.40 (m, 2H), 1.16 (br,
3H), 0.90 (br, 3H). LC-MS: Rt = 2.53 min. (ESI): m/z = 228 [M + H]+.
Ethyl (4-Aminophenyl)phenylphosphinate (64c). According to the
general Pd-catalyzed coupling procedure, 4-bromo-nitrobenzene (2.02
g, 10.0 mmol) and ethyl phenylphosphinate 62c (1.87 g, 11.0 mmol)
were reacted, affording a yellow oil (1.849 g, 64%). 31P NMR (DMSO-
d6, 242 MHz): δ 29.7 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 8.33 (d,
JAABB = 6.1 Hz, 2H), 8.04 (dd, 3JPH ∼ JAABB = 9.5 Hz, 2H), 7.81 (dd, 3JPH
∼ JAABB = 9.2 Hz, 2H), 7.60−7.65 (m, 1H), 7.50−7.65 (m, 2H), 4.00−
4.10 (m, 2H), 1.31 (t, 3JHH = 6.7 Hz, 3H). LC-MS: Rt = 3.49 min. (ESI):
m/z = 292 [M + H]+. Reduction of ethyl (4-nitrophenyl)-
phenylphosphinate (1.809 g, 6.22 mmol) was carried out according to
the nitro group reduction described for compound 26a. The free base
was prepared as a yellow solid (1.435 g, 88%). 31P NMR (DMSO-d6, 242
(DMSO-d6, 300 MHz): δ 9.99 (s, 1H), 8.63 (s, 1H), 7.90−7.93 (m, 1H),
7.70−7.74 (m, 1H), 7.7−7.5 (m, 3H), 7.0−7.2 (m, 3H), 6.83−6.88 (m,
2
1H), 3.87 (s, 3H), 2.72 (d, JPH = 14.4 Hz, 2H), 1.35−1.50 (m, 2H),
1.15−1.3 (m, 2H), 1.04 (t, 3JHH = 8.4 Hz, 3H). LC-MS: Rt = 0.44 and
2.41 min. (ESI): m/z = 397 [M + H]+.
General Method for the Pd-Catalyzed Coupling of H-Phosphi-
nates and Aromatic Halogens. The appropriate halogeno-nitro-
benzene (1 equiv) was dissolved in dry toluene (5 mL/mmol) then the
H-phosphinate (1.1 equiv), triethylamine (4 equiv), and tetrakis
(triphenylphosphine)palladium (0) (0.02 equiv) were added under
inert atmosphere. The reaction mixture was refluxed for 20 h under inert
atmosphere. The mixture was cooled to RT then the precipitate was
filtered off. The filtrate was evaporated to dryness then the residue was
dissolved in EtOAc (25 mL/mmol). This organic layer was washed with
1 M HCl (15 mL/mmol), 2 M NaOH (15 mL/mmol), and with brine
(15 mL/mmol), respectively. Organic phase was dried with MgSO4, and
volatiles were evaporated under reduced pressure. The crude product
was purified by chromatography in hexane:EtOAc (1:1) to 100%
EtOAc. For the exact quantity of reagents, for further purifications,
yields, and analytical data see the exact examples.
Ethyl (3-Aminophenyl)ethylphosphinate (63a). According to the
general Pd-catalyzed coupling procedure, 3-iodo-nitrobenzene (0.996 g,
4.0 mmol) and ethyl ethylphosphinate 62a (0.537 g, 4.4 mmol) were
reacted, giving a yellow oil (0.14 g, 14%). 31P NMR (DMSO-d6, 242
MHz): δ 45.8 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 8.40−8.50 (m,
3
3
3
2H), 8.14 (t, JPH ∼ JHH = 7.7 Hz, 1H), 7.86 (t, JHH = 8.5 Hz, 1H),
3.95−4.00 (m, 1H), 3.80−3.90 (m, 1H), 1.95−2.25 (m, 2H), 1.22 (s,
3H), 0.96 (dt, 3JPH = 19.1 Hz, 3JHH = 9.0 Hz, 3H). LC-MS: Rt = 2.83 min.
(ESI): m/z = 244 [M + H]+. Reduction of ethyl ethyl (3-
nitrophenyl)phosphinate (0.105 g, 0.43 mmol) was carried out
according to the nitro group reduction described for compound 26a.
The free base was prepared as a yellow oil (0.078 g, 85%). 31P NMR
(DMSO-d6, 242 MHz): δ 48.3 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
7.10−7.20 (m, 1H), 6.93 (d, 3JPH = 12.9 Hz, 1H), 6.70−6.88 (m, 2H),
5.34 (br, 2H), 3.85−3.95 (m, 1H), 3.70−3.80 (m, 1H), 1.75−1.85 (m,
2H), 1.19 (t, 3JHH = 6.9 Hz, 3H), 0.94 (dt, 3JPH = 18.5 Hz, 3JHH = 7.4 Hz,
3H). LC-MS: Rt = 2.03 min. (ESI): m/z = 214 [M + H]+.
MHz): δ 33.6 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 7.68 (dd, 3JPH
∼
3JHH = 9.1 Hz, 2H), 7.40−7.60 (m, 3H), 7.37 (dd, 3JPH ∼ JAABB = 9.4 Hz,
2H), 6.60 (d, JAABB = 5.9 Hz, 2H), 5.79 (br, 2H), 3.85−3.95 (m, 2H),
1.24 (br, 3H). LC-MS: Rt = 2.83 min. (ESI): m/z = 262 [M + H]+.
Diethyl (4-Aminophenyl)phosphonate (64d). According to the
general Pd-catalyzed coupling procedure, 4-bromo-nitrobenzene (5.05
g, 25.0 mmol) and diethyl phosphite 62d (3.795 g, 27.5 mmol) were
1
reacted, giving a yellow oil (3.948 g, 61%). H NMR (DMSO-d6, 300
MHz): δ 8.35 (dd, JAABB = 8.7 Hz, 4JPH = 3.1 Hz, 2H), 7.99 (dd, JAABB
=
8.7 Hz, 3JPH = 12.5 Hz, 2H), 4.00−4.10 (m, 4H), 1.25 (t, 3JHH = 7.0 Hz,
6H). LC-MS: Rt = 3.25 min. (ESI): m/z = 260 [M + H]+. Reduction of
diethyl (4-nitrophenyl)phosphonate (3.947 g, 15.2 mmol) was carried
out according to the nitro group reduction described at compound 26a.
The free base was prepared as a yellow solid (1.807 g, 52%). 31P NMR
(DMSO-d6, 242 MHz): δ 20.3 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
7.31 (dd, 3JPH = 12.5 Hz, JAABB = 8.5 Hz, 2H), 6.60 (d, JAABB = 8.5 Hz,
4JPH = 3.7 Hz, 2H), 5.77 (br, 2H), 3.80−4.00 (m, 4H), 1.18 (t, 3JHH = 7.0
Diethyl (3-Aminophenyl)phosphonate (63d). According to the
general Pd-catalyzed coupling procedure, 3-iodo-nitrobenzene (2.49 g,
10.0 mmol) and diethylphosphite 62d (1.518 g, 11.0 mmol) were
reacted, giving a yellow oil (0.951 g, 37%). 31P NMR (DMSO-d6, 242
MHz): δ 13.5 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 8.47 (d, 3JHH
=
8.6 Hz, 1H), 8.40 (d, 3JPH = 14.2 Hz, 1H), 8.14 (dd, 3JPH = 12.3 Hz, 3JHH
= 7.5 Hz, 1H), 7.86 (dt, 3JHH = 8.5 Hz, 4JPH = 4.1 Hz, 1H), 4.00−4.15 (m,
4H), 1.26 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt = 3.20 min. (ESI): m/z =
260 [M + H]+. Reduction of diethyl (3-nitrophenyl)phosphonate
(0.804 g, 3.1 mmol) was carried out according to the nitro group
reduction described for compound 26a. The free base was prepared as a
yellow oil (0.638 g, 90%). 31P NMR (DMSO-d6, 242 MHz): δ 16.4 ppm.
1H NMR (DMSO-d6, 300 MHz): δ 7.25−7.50 (m, 4H), 5.80 (br, 2H),
3.95−4.05 (m, 4H), 1.24 (t, 3JHH = 7.1 Hz, 6H). LC-MS: Rt = 2.34 min.
(ESI): m/z = 230 [M + H]+.
Hz, 6H). LC-MS: Rt = 2.48 min. (ESI): m/z = 230 [M + H]+.
Ethyl Ethyl (3-{[6-(3-Methoxyphenyl)pyrimidin-4-yl]amino}-
phenyl)phosphinate (65). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.057 g,
0.26 mmol) and ethyl (3-aminophenyl)ethylphosphinate·HCl 63a
(0.077 g, 0.31 mmol) were reacted. The crude product was recrystallized
from MeCN to give a yellow powder (0.035 g, 34%); mp = 155 °C
1
(MeCN). 31P NMR (DMSO-d6, 242 MHz): δ 47.7 ppm. H NMR
(DMSO-d6, 300 MHz): δ 9.85 (s, 1H), 8.72 (s, 1H), 7.85−8.10 (m, 3H),
7.40−7.55 (m, 3H), 7.28−7.40 (m, 1H), 7.14−7.25 (m, 1H), 7.02−7.12
(m, 1H), 3.91 (s, 3H), 3.80−4.00 (m, 2H), 1.80−1.95 (m, 2H), 1.23 (br,
3H), 0.97 (dt, 3JPH = 17.3 Hz, 3JHH = 6.4 Hz, 3H). LC-MS: Rt = 2.56 min.
(ESI): m/z = 398 [M + H]+.
Ethyl (4-Aminophenyl)ethylphosphinate (64a). According to the
general Pd-catalyzed coupling procedure, 4-bromo-nitrobenzene (2.02
g, 10.0 mmol) and ethyl ethylphosphinate 62a (1.342 g, 11.0 mmol)
were reacted, yielding a yellow oil (1.312 g, 54%), which upon standing
1
crystallized. 31P NMR (DMSO-d6, 242 MHz): δ 45.7 ppm. H NMR
3959
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Ethyl Ethyl (3-{[6-(4-Nitrophenyl)pyrimidin-4-yl]amino}phenyl)-
phosphinate (66). According to the general coupling procedure, 4-
chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.073 g, 0.31 mmol) and
diethyl (3-aminophenyl)phosphonate·HCl 63d (0.091 g, 0.37 mmol)
were reacted. The crude product was recrystallized from MeCN to give a
yellow powder (0.066 g, 52%); mp = 211 °C (MeCN). 31P NMR
(DMSO-d6, 242 MHz): δ 47.5 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
10.07 (s, 1H), 8.83 (s, 1H), 8.81 (s, 1H), 8.47 (d, 3JHH = 6.6 Hz, 1H),
8.37 (d, 3JHH = 6.9 Hz, 1H), 8.00−8.10 (m, 2H), 7.85 (t, 3JHH = 7.2 Hz,
1H), 7.45−7.55 (m, 1H), 7.30−7.45 (m, 2H), 3.90−4.00 (m, 1H),
(4-aminophenyl)ethylphosphinate·HCl 64a (0.15 g, 0.6 mmol) were
reacted. The crude product was recrystallized from MeCN, providing a
yellow powder (0.135 g, 66%); mp = 241−242 °C (MeCN). 31P NMR
(DMSO-d6, 242 MHz): δ 47.4 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
10.20 (s, 1H), 8.85 (s, 2H), 8.48 (d, 3JHH = 6.3 Hz, 1H), 8.37 (d, 3JHH
=
5.7 Hz, 1H), 7.30−7.70 (m, 3H), 7.60−7.70 (m, 2H),7.40−7.50 (m,
1H), 3.90−3.93 (m, 1H), 3.76−3.79 (m, 1H), 1.83−1.87 (m, 2H), 1.20
(br, 3H), 0.96 (dt, 3JPH = 18.3 Hz, 3JHH = 9.3 Hz, 3H). LC-MS: Rt = 3.57
min. (ESI): m/z = 413 [M + H]+.
Ethyl (4-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl)-
propylphosphinate (69b). According to the general coupling
procedure, 4-chloro-6- 3-nitrophenyl)-pyrimidine 13b (0.118 g, 0.5
mmol) and ethyl (4-aminophenyl)propylphosphinate·HCl 64b (0.167
g, 0.6 mmol) were reacted. The crude product was recrystallized from
MeCN, providing a yellow powder (0.059 g, 28%); mp = 207−209 °C
3.80−3.90 (m, 1H), 1.80−2.00 (m, 2H), 1.23 (br, 3H), 0.99 (dt, 3JPH
=
18.3 Hz, 3JHH = 8.7 Hz, 3H). LC-MS: Rt = 3.55 min. (ESI): m/z = 413
[M + H]+.
Ethyl (3-{[6-(4-Aminophenyl)pyrimidin-4-yl]amino}phenyl)-
ethylphosphinate (67). Reduction of the nitro compound 66 (0.048
g, 0.12 mmol) was carried out according to the nitro group reduction
described for compound 26a, giving a yellow powder (0.04 g, 80%); mp
= 213-214 °C (EtOH, Et2O). 31P NMR (DMSO-d6, 242 MHz): δ 47.2
ppm. 1H NMR (DMSO-d6, 300 MHz): δ 11.06 (s, 1H), 8.87 (s, 1H),
8.00−8.15 (m, 2H), 7.75−7.85 (m, 2H), 7.55−7.62 (m, 2H), 7.38−7.50
(m, 3H), 5.30 (br, 2H), 3.95−4.00 (m, 1H), 3.80−3.85 (m, 1H), 1.88−
1.93 (m, 2H), 1.23 (br, 3H), 0.98 (dt, 3JPH = 18.7 Hz, 3JHH = 6.8 Hz, 3H).
LC-MS: Rt = 2.37 min. (ESI): m/z = 383 [M + H]+.
1
(MeCN). 31P NMR (DMSO-d6, 242 MHz): δ 45.9 ppm. H NMR
(DMSO-d6, 300 MHz): δ 10.20 (s, 1H), 8.85 (s, 2H), 8.48 (d, 3JHH = 6.5
3
Hz, 1H), 8.37 (d, JHH = 7.3 Hz, 1H), 7.75−7.95 (m, 3H), 7.65−7.75
(m, 2H), 7.48 (s, 1H), 3.90−3.95 (m, 1H), 3.75−3.80 (m, 1H), 1.83−
1.89 (m, 2H), 1.40−1.45 (m, 2H), 1.20 (br, 3H), 0.92 (br, 3H). LC-MS:
Rt = 3.87 min. (ESI): m/z = 427 [M + H]+.
Ethyl (4-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl)-
phenylphosphinate (69c). According to the general coupling
procedure, 4-chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.118 g, 0.5
mmol) and ethyl (4-aminophenyl)phenylphosphinate·HCl 64c (0.179
g, 0.6 mmol) were reacted. The crude product was recrystallized from
MeCN, providing a pale-yellow powder (0.149 g, 65%); mp = 243 °C
Ethyl Ethyl (4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}-
phenyl)phosphinate (68a). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.11 g, 0.5
mmol) and ethyl (4-aminophenyl)ethylphosphinate·HCl 64a (0.15 g,
0.6 mmol) were reacted. The crude product was purified by preparative
TLC with 100% EtOAc and 1 v/v% AcOH as eluent. The pure product
was extracted with MeOH from the silica. After evaporation, 68a was
obtained as a yellow powder (0.075 g, 38%); mp = 151−153 °C
1
(MeCN). 31P NMR (DMSO-d6, 242 MHz): δ 32.1 ppm. H NMR
(DMSO-d6, 300 MHz): δ 10.37 (s, 1H), 8.84 (s, 2H), 8.47 (d, 3JHH = 5.7
3
Hz, 1H), 8.36 (d, JHH = 6.3 Hz, 1H), 7.90−8.00 (m, 2H), 7.60−7.90
(m, 5H), 7.40−7.60 (m, 4H), 3.80−4.05 (m, 2H), 1.30 (t, 3JHH = 7.5 Hz,
1
(MeOH). 31P NMR (DMSO-d6, 242 MHz): δ 47.3 ppm. H NMR
3H). LC-MS: Rt = 4.02 min. (ESI): m/z = 461 [M + H]+.
(DMSO-d6, 600 MHz): δ 9.95 (s, 1H), 8.74 (s, 1H), 7.94 (dd, 3JHH = 7.8
Hz, 4JHH = 2.0 Hz, 1H), 7.88 (dd, JAABB = 8.4 Hz, 4JPH = 2.4 Hz, 2H), 7.65
(dd, JAABB = 8.4 Hz, 3JPH = 10.8 Hz, 2H), 7.51 (s, 1H), 7.44 (t, 3JHH = 7.2
Hz, 1H), 7.17 (d, 3JHH = 8.4 Hz, 1H), 7.06 (t, 3JHH = 7.2 Hz, 1H), 3.88 (s,
3H), 3.85−3.92 (m, 1H), 3.70−3.78 (m, 1H), 1.77−1.90 (m, 2H), 1.17
(t, 3JHH = 7.2 Hz, 3H), 0.93 (dt, 3JPH = 18.6 Hz, 3JHH = 7.8 Hz, 3H). LC-
MS: Rt = 2.60 min. (ESI): m/z = 398 [M + H]+.
Ethyl (4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl)-
ethylphosphinate (70a). Reduction of the nitro compound 69a
(0.108 g, 0.26 mmol) was carried out according to the nitro group
reduction described for compound 26a, giving a yellow powder (0.084 g,
77%); mp = 136−138 °C (EtOH, Et2O). 31P NMR (DMSO-d6, 242
MHz): δ 47.7 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 10.88 (s, 1H),
8.87 (s, 1H), 7.92−7.96 (m, 2H), 7.83−7.87 (m, 2H), 7.70−7.75 (m,
2H), 7.59−7.62 (m, 1H), 7.42−7.47 (m, 2H), 4.00 (br, 2H), 3.90−4.00
(m, 1H), 3.80−3.90 (m, 1H), 1.85−1.89 (m, 2H), 1.21 (br, 3H), 0.96
(dt, 3JPH = 18.5 Hz, 3JHH = 8.0 Hz, 3H). LC-MS: Rt = 2.23 and 2.39 min.
(ESI): m/z = 383 [M + H]+.
Ethyl (4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl)-
propylphosphinate (70b). Reduction of the nitro compound 69b
(0.039 g, 0.09 mmol) was carried out according to the nitro group
reduction described for compound 26a, affording a yellow powder
(0.035 g, 90%); mp = 140−142 °C (EtOH, DEE). 31P NMR (DMSO-
d6, 242 MHz): δ 45.7 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 10.75 (s,
1H), 8.87 (s, 1H), 7.90−7.93 (m, 2H), 7.69−7.82 (m, 4H), 7.55−7.60
(m, 1H), 7.35−7.44 (m, 2H), 3.68 (br, 4H), 1.83−1.87 (m, 2H), 1.40−
1.44 (m, 2H), 1.21 (t, 3JHH = 7.0 Hz, 3H), 0.92 (t, 3JHH = 7.0 Hz, 3H).
LC-MS: Rt = 2.61 min. (ESI): m/z = 397 [M + H]+.
Ethyl (4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl)-
phenylphosphinate (70c). Reduction of the nitro compound 69c
(0.132 g, 0.29 mmol) was carried out according to the nitro group
reduction described for compound 26a, giving a yellow powder (0.073 g,
59%); mp = 131−134 °C (EtOH, Et2O). 31P NMR (DMSO-d6, 242
MHz): δ 33.0 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 9.98 (s, 1H),
8.73 (s, 1H), 7.99 (d, 3JHH = 5.4 Hz, 1H), 7.71−7.76 (m, 5H), 7.55−7.60
(m, 3H), 7.10−7.35 (m, 4H), 6.70−6.75 (m, 1H), 5.29 (br, 2H), 3.97−
4.01 (m, 2H), 1.29 (t, 3JHH = 6.7 Hz, 3H). LC-MS: Rt = 2.81 min. (ESI):
m/z = 431 [M + H]+.
Ethyl (4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl)-
propylphosphinate (68b). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.11 g, 0.5
mmol) and ethyl (4-aminophenyl)propylphosphinate·HCl 64b (0.132
g, 0.6 mmol) were reacted. The crude product was purified by
preparative TLC with 100% EtOAc as eluent. The product was extracted
with MeOH from the silica. After evaporation, 68b was isolated as a
yellow powder (0.041 g, 20%); mp = 85 °C (MeOH). 31P NMR
(DMSO-d6, 242 MHz): δ 45.7 ppm. 1H NMR (DMSO-d6, 600 MHz): δ
9.94 (s, 1H), 8.74 (s, 1H), 7.94 (dd, 3JHH = 7.8 Hz, 4JHH = 1.2 Hz, 1H),
7.88 (dd, JAABB = 8.4 Hz, 4JPH = 2.4 Hz, 2H), 7.65 (dd, JAABB = 9.0 Hz,
3JPH = 10.8 Hz, 2H), 7.51 (s, 1H), 7.44 (t, 3JHH = 7.8 Hz, 1H), 7.17 (d,
3JHH = 8.4 Hz, 1H), 7.06 (t, 3JHH = 7.8 Hz, 1H), 3.88 (s, 3H), 3.84−3.90
(m, 1H), 3.68−3.76 (m, 1H), 1.76−1.88 (m, 2H), 1.30−1.48 (m, 2H),
1.17 (t, 3JHH = 7.2 Hz, 3H), 0.89 (t, 3JHH = 7.8 Hz, 3H). LC-MS: Rt =
2.82 min. (ESI): m/z = 412 [M + H]+.
Ethyl (4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl)-
phenylphosphinate (68c). According to the general coupling
procedure, 4-chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.11 g, 0.5
mmol) and ethyl (4-aminophenyl)phenylphosphinate·HCl 64c (0.179
g, 0.6 mmol) were reacted. The crude product was recrystallized from
MeCN, providing a pale-yellow powder (0.129 g, 58%); mp = 197−198
°C (MeCN). 31P NMR (DMSO-d6, 242 MHz): δ 32.0 ppm. 1H NMR
(DMSO-d6, 300 MHz): δ 9.97 (s, 1H), 8.77 (s, 1H), 7.96 (d, 3JHH = 7.4
Hz, 1H), 7.85−7.95 (m, 2H), 7.70−7.80 (m, 4H), 7.40−7.60 (m, 5H),
7.19 (d, 3JHH = 7.7 Hz, 1H), 7.09 (t, 3JHH = 7.2 Hz, 1H), 3.90−3.80 (m,
2H), 3.90 (s, 3H), 1.29 (t, 3JHH = 7.0 Hz, 3H). LC-MS: Rt = 3.06 min.
(ESI): m/z = 446 [M + H]+.
Diethyl (3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl)-
phosphonate (71). According to the general coupling procedure, 4-
chloro-6-(2-methoxyphenyl)-pyrimidine 13a (0.121 g, 0.55 mmol) and
diethyl (3-aminophenyl)phosphonate·HCl 63d (0.218 g, 0.82 mmol)
were reacted. The crude product was suspended in diisoprpylether, and
filtration gave a pale-yellow powder (0.132 g, 58%); mp = 122−123 °C
Ethyl Ethyl (4-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl)-
phosphinate (69a). According to the general coupling procedure, 4-
chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.118 g, 0.5 mmol) and ethyl
1
(DIPE). 31P NMR (DMSO-d6, 242 MHz): δ 17.0 ppm. H NMR
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(DMSO-d6, 300 MHz): δ 9.87 (s, 1H), 8.74 (s, 1H), 8.10 (d, 3JPH = 15.4
Hz, 1H), 8.06 (d, 3JHH = 8.4 Hz, 1H), 7.97 (d, 3JHH = 7.5 Hz, 1H), 7.44−
7.55 (m, 3H), 7.32 (dd, 3JPH = 12.6 Hz, 3JHH = 7.4 Hz, 1H), 7.19 (d, 3JHH
= 8.3 Hz, 1H), 7.09 (t, 3JHH = 7.4 Hz, 1H), 3.98−4.09 (m, 4H), 3.91 (s,
3H), 1.26 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt = 2.70 min. (ESI): m/z =
414 [M + H]+.
31P NMR (DMSO-d6, 242 MHz): δ 11.6 ppm. H NMR (DMSO-d6,
300 MHz): δ 10.16 (s, 1H), 8.82 (s, 2H), 8.43 (d, 3JHH = 7.7 Hz, 1H),
1
8.38 (d, 3JHH = 8.1 Hz, 1H), 8.00 (d, 3JPH = 14.7 Hz, 1H), 7.94 (d, 3JHH
=
7.8 Hz, 1H), 7.86 (t, 3JHH = 8.0 Hz, 1H), 7.33−7.49 (m, 3H), 5.27 (br,
2H). LC-MS: Rt = 2.50 min. (ESI): m/z = 373 [M + H]+.
(4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl)-
phosphonic Acid (78). 78 was prepared with the method described for
compound 45a starting from diethyl phosphonate 73 (0.077 g, 0.19
mmol). The reaction yielded a yellow powder (0.015 g, 22%); mp> 350
°C (EtOH). 31P NMR (DMSO-d6, 242 MHz): δ 10.7 ppm. 1H NMR
(DMSO-d6, 300 MHz): δ 9.85 (s, 1H), 8.74 (s, 1H), 7.90 (d, 3JHH = 6.5
Hz, 1H), 7.52−7.80 (m, 4H), 7.40−7.50 (m, 2H), 7.19 (d, 3JHH = 8.0
Hz, 1H), 7.09 (dd, 3JHH = 7.0 Hz, 3JHH = 6.6 Hz, 1H), 4.60 (br, 2H), 3.91
(s, 3H). LC-MS: Rt = 0.46 and 1.83 min. (ESI): m/z = 358 [M + H]+.
(4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl)phosphonic
Acid (79). 79 was prepared with the method described at compound 45a
starting from diethyl phosphonate 75 (0.086 g, 0.22 mmol). The
reaction yielded a yellow powder (0.046 g, 61%); mp > 350 °C (EtOH).
Diethyl (3-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl)-
phosphonate (72). According to the general coupling procedure, 4-
chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.249 g, 1.06 mmol) and
diethyl (3-aminophenyl)phosphonate·HCl 63d (0.421 g, 1.59 mmol)
were reacted. The crude product was suspended in DIPE, and filtration
gave a yellow powder (0.364 g, 80%); mp = 206−208 °C (DIPE). 31P
1
NMR (DMSO-d6, 242 MHz): δ 16.9 ppm. H NMR (DMSO-d6, 300
MHz): δ 10.04 (s, 1H), 8.84 (s, 1H), 8.82 (s, 1H), 8.47 (d, 3JHH = 7.8 Hz,
1H), 8.37 (d, 3JHH = 7.2 Hz, 1H), 8.13 (d, 3JPH = 14.7 Hz, 1H), 8.04 (d,
3JHH = 8.1 Hz, 1H), 7.86 (t, 3JHH = 8.1 Hz, 1H), 7.53 (dt, 3JHH = 7.8 Hz,
4JPH = 5.2 Hz, 1H), 7.40 (s, 1H), 7.19 (dd, 3JPH = 12.9 Hz, 3JHH = 7.4 Hz,
1H), 4.00−4.10 (m, 4H), 1.26 (t, 3JHH = 6.9 Hz, 6H). LC-MS: Rt = 3.85
min. (ESI): m/z = 429 [M + H]+.
31P NMR (DMSO-d6, 242 MHz): δ 12.0 ppm. H NMR (DMSO-d6,
1
300 MHz): δ 10.63 (s, 1H), 8.83 (s, 1H), 7.83 (dd, JAABB = 8.4 Hz, 4JPH
=
Diethyl (4-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl)-
phosphonate (73). According to the general coupling procedure, 4-
chloro-6- (2-methoxyphenyl)-pyrimidine 13a (0.097 g, 0.44 mmol) and
diethyl (4-aminophenyl)phosphonate·HCl 64d (0.174 g, 0.66 mmol)
were reacted. The crude product was suspended in DIPE, and filtration
gave a yellow powder (0.106 g, 58%); mp = 151 °C (DIPE). 31P NMR
(DMSO-d6, 242 MHz): δ 17.9 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.97 (s, 1H), 8.78 (s, 1H), 7.98 (dd, 3JHH = 7.7 Hz, 4JHH = 1.5 Hz, 1H),
7.92 (dd, JAABB = 8.5 Hz, 4JPH = 3.5 Hz, 2H), 7.67 (dd, 3JPH = 12.6 Hz,
JAABB = 8.5 Hz, 2H), 7.55 (s, 1H), 7.47 (dd, 3JHH = 7.7 Hz, 3JHH = 7.2 Hz,
1H), 7.19 (d, 3JHH = 8.3 Hz, 1H), 7.09 (t, 3JHH = 7.2 Hz, 1H), 3.94−4.05
(m, 4H), 3.92 (s, 3H), 1.24 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt = 2.82
min. (ESI): m/z = 414 [M + H]+.
2.7 Hz, 2H), 7.64−7.72 (m, 4H), 7.51 (t, 3JHH = 7.9 Hz, 1H), 7.37 (s,
1H), 7.26 (t, 3JHH = 7.5 Hz, 1H), 5.70 (br, 4H). LC-MS: Rt = 0.45 and
1.07 min. (ESI): m/z = 343 [M + H]+.
General Method for the Suzuki Coupling. To the solution of 4,6-
dichloropyridine (0.373 g; 2.5 mmol) in dimethoxyethane (40 mL)
tetrakis (triphenylphosphine)palladium (0) (0.145 g; 0.13 mmol; 0.05
equiv) was added under inert atmosphere. The mixture was stirred at RT
for 1.5 h then the boronic acid or the boronic acid pinacolate ester (2.5
mmol; 1 equiv), Na2CO3 (0.53 g; 5.0 mmol; 2 equiv), and 0.5 mL of
degassed water were added. The reaction mixture was refluxed for 20 h
under inert atmosphere. Volatiles were evaporated under reduced
pressure then the residue was suspended in 1 M NaH2PO4. The
suspension was extracted 3 times with EtOAc, combined organics were
washed with satd Na2CO3 and with brine and dried with MgSO4. After
evaporation of the volatiles under reduced pressure, the crude product
was purified by column chromatography in hexane:EtOAc (4:1). For
further purifications, yields, and analytical data see the exact examples.
4-Chloro-6-(2-ethoxyphenyl)pyrimidine (13c). Yellow crystals
(0.267 g, 46%); mp = 75−76 °C (EtOAc, hexane). 1H NMR
(DMSO-d6, 300 MHz): δ 7.5−7.7 (m, 4H), 7.3−7.4 (m, 1H), 6.9−
7.0 (m, 1H), 4.09 (q, 3JHH = 6.9 Hz, 2H), 1.36 (t, 3JHH = 6.7 Hz, 3H). LC-
MS: Rt = 4.31 min. (ESI): m/z = 234 [M + H]+.
Diethyl (4-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl)-
phosphonate (74). According to the general coupling procedure, 4-
chloro-6-(3-nitrophenyl)-pyrimidine 13b (0.25 g, 1.06 mmol) and
diethyl (4-aminophenyl)phosphonate·HCl 64d (0.422 g, 1.59 mmol)
were reacted. The crude product was suspended in Et2O, and precipitate
was filtered. Recrystallization from MeCN gave a yellow powder (0.209
g, 46%); mp = 219 °C (MeCN). 31P NMR (DMSO-d6, 242 MHz): δ
1
17.7 ppm. H NMR (DMSO-d6, 300 MHz): δ 10.30 (s, 1H), 8.85 (s,
2H), 8.48 (d, 3JHH = 7.7 Hz, 1H), 8.37 (d, 3JHH = 7.9 Hz, 1H), 7.94 (dd,
JAABB = 8.2 Hz, 4JPH = 3.1 Hz, 2H), 7.85 (t, 3JHH = 8.0 Hz, 1H), 7.68 (dd,
3JPH = 12.5 Hz, JAABB = 8.4 Hz, 2H), 7.51 (s, 1H), 3.94−4.05 (m, 4H),
4-Chloro-6-(2-propoxyphenyl)pyrimidine (13d). White crystals
(0.159 g, 26%). H NMR (DMSO-d6, 300 MHz): δ 9.1 (s, 1H), 8.19
1.24 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt = 3.87 min. (ESI): m/z = 429 [M
+ H]+.
1
(s, 1H), 8.01 (d, 3JHH = 5.8 Hz, 1H), 7.53 (s, 1H), 7.21 (d, 3JHH = 6.6 Hz,
1H), 7.12 (s, 1H), 4.1 (t, 3JHH = 7.0 Hz, 2H), 1.73−1.8 (m, 2H), 1.02 (t,
3JHH = 7.3 Hz, 3H). LC-MS: Rt = 4.68 min. (ESI): m/z = 248 [M + H]+.
4-Chloro-6-(2-isopropoxyphenyl)pyrimidine (13e). Colorless liquid
(0.258 g, 42%). 1H NMR (DMSO-d6, 300 MHz): δ 9.09 (s, 1H), 8.17 (s,
1H), 8.01 (d, 3JHH = 7.1 Hz, 1H), 7.52 (t, 3JHH = 7.0 Hz, 1H), 7.23 (d,
3JHH = 7.2 Hz, 1H), 7.1 (t, 3JHH = 7.1 Hz, 1H), 3.42−3.51 (m, 1H), 1.32
(d, 3JHH = 6.9 Hz, 6H). LC-MS: Rt = 4.55 min. (ESI): m/z = 248 [M +
H]+.
Diethyl (4-{[6-(3-Aminophenyl)pyrimidin-4-yl]amino}phenyl)-
phosphonate (75). Reduction of the nitro compound 74 (0.14 g,
0.33 mmol) was carried out according to the nitro group reduction
described for compound 26a. The free base was prepared as a yellow
powder (0.127 g, 97%); mp = 174−175 °C (EtOH). 31P NMR (DMSO-
d6, 242 MHz): δ 17.8 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 9.99 (s,
1H), 8.75 (s, 1H), 7.91 (dd, JAABB = 8.5 Hz, 4JPH = 3.5 Hz, 2H), 7.67 (dd,
3JPH = 12.5 Hz, JAABB = 8.5 Hz, 2H), 7.31 (s, 1H), 7.23 (s, 1H), 7.10−
7.20 (m, 2H), 6.65−6.71 (m, 1H), 5.30 (br, 2H; 3.94−4.04 (m, 4H),
1.23 (t, 3JHH = 7.0 Hz, 6H). LC-MS: Rt = 2.56 min. (ESI): m/z = 399 [M
+ H]+.
4-Chloro-6-(2-phenoxyphenyl)pyrimidine (13f). White crystals
1
(0.44 g, 63%); mp = 73 °C (EtOAc, hexane), H NMR (DMSO-d6,
300 MHz): δ 9.12 (s, 1H), 8.08 (s, 1H), 8.03 (d, 3JHH = 7.3 Hz, 1H), 7.54
(t, 3JHH = 6.8 Hz, 1H), 7.3−7.5 (m, 3H), 7.16 (t, 3JHH = 6.7 Hz, 1H),
7.0−7.1 (m, 3H). LC-MS: Rt = 4.73 min. (ESI): m/z = 282 [M + H]+.
4-[2-(Benzyloxy)phenyl]-6-chloropyrimidine (13g). White crystals
(0.388 g, 52%); mp = 87−88 °C (EtOAc, hexane), 1H NMR (DMSO-
d6, 300 MHz): δ 9.1 (s, 1H), 8.19 (s, 1H), 8.01 (d, 3JHH = 6.8 Hz, 1H),
7.3−7.6 (m, 7H), 7.15 (t, 3JHH = 5.6 Hz, 1H), 5.27 (s, 2H). LC-MS: Rt =
4.74 min. (ESI): m/z = 296 [M + H]+.
(3-{[6-(2-Methoxyphenyl)pyrimidin-4-yl]amino}phenyl)-
phosphonic Acid (76). 76 was prepared with the method described for
compound 45a starting from diethyl phosphonate 71 (0.05 g, 0.12
mmol). The reaction yielded a yellow powder (0.03 g, 69%); mp = 250−
253 °C (dec, EtOH). 31P NMR (DMSO-d6, 242 MHz): δ 10.9 ppm. 1H
NMR (DMSO-d6, 300 MHz): δ 11.41 (s, 1H), 8.91 (s, 1H), 8.00 (d, 3JPH
= 14.0 Hz, 1H), 7.91 (d, 3JHH = 4.9 Hz, 1H), 7.77 (d, 3JHH = 7.4 Hz, 1H),
7.61 (t, 3JHH = 7.6 Hz, 1H), 7.40−7.55 (m, 2H), 7.45 (s, 1H), 7.27 (d,
3JHH = 8.2 Hz, 1H), 7.17 (t, 3JHH = 7.4 Hz, 1H), 5.75 (br, 2H), 3.91 (s,
4-Chloro-6-(3-methoxyphenyl)pyrimidine (13h). White crystals
1
3H). LC-MS: Rt = 0.47 and 1.92 min. (ESI): m/z = 358 [M + H]+.
(3-{[6-(3-Nitrophenyl)pyrimidin-4-yl]amino}phenyl)phosphonic
Acid (77). 77 was prepared with the method described for compound
45a starting from diethyl phosphonate 72 (0.15 g, 0.35 mmol). The
reaction yielded a yellow powder (0.039 g, 30%); mp> 350 °C (EtOH).
(0.34 g, 62%). H NMR (CDCl3, 500 MHz): δ 9.02 (s, 1H), 7.23 (s,
1H), 7.65 (s, 1H), 7.61 (d, 3JHH = 8 Hz, 1H), 7.42 (t, 3JHH = 8.0 Hz, 1H),
7.08 (d, JHH = 6.5 Hz, 1H), 3.90 (s, 3H).
4-Chloro-6-(4-methoxyphenyl)pyrimidine (13i). White crystals
(0.21 g, 38%). H NMR (CDCl3, 500 MHz): δ 8.96 (s, 1H), 8.06 (d,
1
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JHH = 8.5 Hz, 2H), 7.67 (s, 1H), 7.02 (d, JHH = 8.5 Hz, 2HAR), 3.89 (s,
3H).
1C), 160.7 (s, 1C), 158.4 (s, 1C), 155.9 (s, 1C), 139.0 (d, JPC = 2.7 Hz,
1C), 133.6 (d, JPC = 7.1 Hz, 1C), 131.0 (d, JPC = 8.2 Hz, 2C), 129.5 (d,
JPC = 2.6 Hz, 1C), 127.6 (s, 1C), 125.7 (d, JPC = 5.6 Hz, 1C), 123.4 (d,
JPC = 5.5 Hz, 1C), 121.0 (s, 1C), 120.4 (d, JPC = 3.0 Hz, 1C), 114.3 (s,
4-Chloro-6-(2,3-dimethoxyphenyl)pyrimidine (13j). White crystals
1
(0.24 g, 38%). H NMR (CDCl3, 500 MHz): δ 9.05 (s, 1H), 8.02 (s,
1H), 7.52 (d, JHH = 6.5 Hz, 1H), 7.19 (t, JHH = 8 Hz, 1H), 7.07 (d, JHH = 8
1C), 105.1 (s, 1C), 70.9 (s, 1C), 60.6 (d, JPC = 6.6 Hz, 1C), 36.4 (d, JPC =
Hz, 1H), 3.93 (s, 3H), 3.80 (s, 3H).
82.9 Hz, 1C), 29.2 (s, 1C), 22.1 (s, 1C), 16.6 (d, JPC = 5.5 Hz, 1C), 15.5
(d, JPC = 5.8 Hz, 1C), 15.4 (d, JPC = 5.5 Hz, 1C). LC-MS: Rt = 3.05 min;
(ESI): m/z = 454 [M + H]+.
4-Chloro-6-(4-fluoro-2-methoxyphenyl)pyrimidine (13k). White
1
crystals (0.12 g, 20%). H NMR (CDCl3, 500 MHz): δ 9.00 (s, 1H),
8.13 (t, JHH = 7 Hz, 1H), 8.05 (s, 1H), 6.82 (t, JHH = 8 Hz, 1H), 6.75 (d,
JHH = 8.5 Hz, 1H), 3.94 (s, 3H).
Ethyl (3-{[6-(2-Phenoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (83). According to the general coupling procedure,
4-chloro-6-(2-phenoxyphenyl)-pyrimidine 13f (0.23 g, 0.81 mmol) and
ethyl (3-aminophenyl)propylphosphinate·HCl 48 (0.236 g, 0.89 mmol)
were reacted. The crude product was purified by chromathography in
hexane:EtOAc (9:1). After evaporation of the fractions under reduced
pressure, the product was crystallized from MeCN to give white crystals
4-[3-(Benzyloxy)phenyl]-6-chloropyrimidine (13l). Yellow oil
(0.592 g, 80%). 1H NMR (CDCl3, 300 MHz): δ 9.09 (s, 1H), 8.36 (s,
1H), 7.8−7.92 (m, 2H), 7.27.7.55 (m, 6H), 7.24 (d, JHH = 7.2 Hz, 1H),
5.22 (s, 2H). LC-MS: Rt = 4.79 min. (ESI): m/z = 296 [M + H]+.
4-Chloro-6-(2-ethylphenyl)pyrimidine (13m). White crystals were
prepared (0.058 g, 11%). 1H NMR (CDCl3, 500 MHz): δ 9.12 (s, 1H),
7.24−7.49 (m, 5H), 2.68−2.78 (m, 2H), 1.08 (t, JHH = 7.5 Hz, 3H).
Ethyl (3-{[6-(2-Ethoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (80). According to the general coupling procedure,
4-chloro-6-(2-ethoxyphenyl)-pyrimidine 13c (0.262 g, 1.12 mmol) and
ethyl (3-aminophenyl)propylphosphinate·HCl 48 (0.326 g, 1.23 mmol)
were reacted. The crude product was purified by chromathography in
hexane:EtOAc (9:1). Evaporation of the fractions under reduced
pressure provided a pale-yellow oil (0.05 g, 10%). 31P NMR (CDCl3,
121 MHz): δ 54.2 ppm. 1H NMR (CDCl3, 500 MHz): δ 8.76 (s, 1H),
7.98 (q, JHH = 4 Hz, 1H), 7.41−7.46 (m, 3H), 7.27−7.38 (m, 3H), 7.06
(t, JHH = 7 Hz, 2H), 6.95 (d, JHH = 8.5 Hz, 1H), 4.01−4.06 (m, 2H),
3.94−4.01 (m, 2H), 3.12 (d, JPH = 16.5 Hz, 2H), 1.59−1.63 (m, 4H),
1.33 (t, JHH = 7.0 Hz, 3H), 1.24 (t, JHH = 7.0 Hz, 3H), 0.96 (t, JHH = 7 Hz,
3H). 13C NMR (CDCl3, 75 MHz): δ 162.0 (s, 1C), 161.0 (s, 1C), 158.5
(s, 1C), 157.2 (s, 1C), 139.2 (d, JPC = 2.6 Hz, 1C), 133.6 (d, JPC = 7.1 Hz,
1C), 131.3 (s, 1C), 131.0 (s, 1C), 129.7 (d, JPC = 2.6 Hz, 1C), 126.8 (s,
1C), 125.8 (d, JPC = 5.5 Hz, 1C), 123.5 (d, JPC = 5.5 Hz, 1C),15.0 (s, 1C),
121.0 (s, 1C), 120.4 (d, JPC = 9.3 Hz, 1C), 112.6 (s, 1C), 105.4 (s, 1C),
64.3 (s, 1C), 60.8 (d, JPC = 6.7 Hz, 1C), 36.6 (d, JPC = 83.0 Hz, 1C), 29.9
(d, JPC = 92.0 Hz, 1C), 16.8 (d, JPC = 5.6 Hz, 1C), 15.6 (s, 1C), 15.5 (s,
1C). LC-MS: Rt = 2.91 min. (ESI): m/z = 440 [M + H]+.
Ethyl (3-{[6-(2-Propoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (81). According to the general coupling procedure,
4-chloro-6-(2-propoxyphenyl)-pyrimidine 13d (0.15 g, 0.6 mmol) and
ethyl (3-aminophenyl)propylphosphinate·HCl 48 (0.175 g, 0.66 mmol)
were reacted. The crude product was purified by chromathography in
hexane:EtOAc (9:1). Evaporation of the fractions under reduced
pressure provided a pale-yellow oil (0.1 g, 37%). 31P NMR (CDCl3, 121
MHz): δ 52.5 ppm. 1H NMR (CDCl3, 500 MHz): δ 8.74 (s, 1H), 7.90−
7.95 (m, 2H), 7.41 (s, 1H), 7.27−7.39 (m, 4H), 7.02 (t, JHH = 8 Hz, 2H),
6.92 (d, JHH = 8.5 Hz, 1H), 3.91−4.18 (m, 4H), 3.11 (d, JPH = 16.5 Hz,
2H), 1.45−1.79 (m, 6H), 1.21 (t, JHH = 7.0 Hz, 3H), 0.93 (t, JHH = 7.5
Hz, 3H), 0.87 (t, JHH = 7.0 Hz, 3H). 13C NMR (CDCl3, 75 MHz): δ
1
(0.12 g, 30%). 31P NMR (CDCl3, 121 MHz): δ 53.3 ppm. H NMR
(CDCl3, 500 MHz): δ 8.73 (s, 1H), 8.25 (s, 1H), 8.03 (d, JHH = 6.5 Hz,
1H), 7.40 (s, 1H), 7.21−7.35 (m, 6H), 7.14 (t, JHH = 9.0 Hz, 1H), 7.06
(t, JHH = 7.5 Hz, 1H), 6.97 (d, JHH = 7.5 Hz, 1H), 6.92 (d, JHH = 8.0 Hz,
3H), 3.94−4.08 (m, 2H), 3.01 (d, JPH = 16.5 Hz, 2H), 1.59−1.65 (m,
4H), 1.22 (t, JHH = 7.3 Hz, 3H), 0.94 (t, JHH = 7.0 Hz, 3H). 13C NMR
(CDCl3, 75 MHz): δ 160.9 (s, 1C), 160.8 (s, 1C), 158.5 (s, 1C), 156.9
(s, 1C), 154.9 (s, 1C), 139.2 (d, JPC = 2.7 Hz, 1C), 133.2 (d, JPC = 7.1 Hz,
1C), 131.1 (d, JPC = 3.6 Hz, 1C), 129.9 (s, 1C), 129.7 (s, 1C), 129.5 (s,
1C), 129.4 (s, 1C), 125.3 (d, JPC = 5.4 Hz, 1C), 124.1 (s, 1C), 123.5 (s,
1C), 122.8 (d, JPC = 5.6 Hz, 2C), 120.0 (d, JPC = 2.9 Hz, 2C), 119.6 (s,
1C), 118.6 (s, 1C), 105.4 (s, 1C), 60.7 (d, JPC = 6.7 Hz, 1C), 36.4 (d, JPC
= 83.0 Hz, 1C), 29.8 (d, JPC = 92.0 Hz, 1C), 16.7 (d, JPC = 5.6 Hz, 1C),
15.6 (d, JPC = 8.6 Hz, 1C), 15.5 (d, JPC = 2.7 Hz, 1C). LC-MS: Rt = 3.48
min. (ESI): m/z = 488 [M + H]+.
Ethyl [3-({6-[2-(Benzyloxy)phenyl]pyrimidin-4-yl}amino)benzyl]-
propylphosphinate (84). According to the general coupling procedure,
4-[2-(benzyloxy)phenyl]-6-chloropyrimidine 13g (0.369 g, 1.25 mmol)
and ethyl (3-aminophenyl)propylphosphinate·HCl 48 (0.363 g, 1.37
mmol) were reacted. The crude product was purified by chromathog-
raphy in hexane:EtOAc (9:1). After evaporation of the fractions under
reduced pressure, the product was crystallized from Et2O to give white
crystals (0.23 g, 37%). 31P NMR (CDCl3, 121 MHz): δ 52.5 ppm. 1H
NMR (CDCl3, 500 MHz): δ 8.74 (s, 1H), 8.24 (d, JHH = 4.5 Hz, 1H),
7.87 (d, JHH = 7.5 Hz, 1H), 7.37 (s, 1H), 7.19−7.33 (m, 8H), 7.09 (t, J =
8.0 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 6.92−6.97 (m, 2H), 5.05 (s, 2H),
3.89−4.01 (m, 2H), 3.00 (d, JPH = 16.5 Hz, 2H), 1.42−1.59 (m, 4H),
1.17 (t, JHH = 7.5 Hz, 3H), 0.89 (t, JHH = 6.5 Hz, 3H). 13C NMR (CDCl3,
75 MHz): δ 161.6 (s, 1C), 160.7 (s, 1C), 158.3 (s, 1C), 156.6 (s, 1C),
139.3 (d, JPC = 2.6 Hz, 1C), 136.7 (s, 1C), 133.2 (d, JPC = 7.1 Hz, 1C),
130.9 (d, JPC = 5.4 Hz, 1C), 129.4 (d, JPC = 2.5 Hz, 1C), 128.6 (s, 1C),
127.9 (s, 2C), 127.6 (s, 1C), 127.1 (s, 1C), 125.1 (d, JPC = 5.4 Hz, 2C),
122.6 (d, JPC = 5.6 Hz, 1C), 121.4 (s, 1C), 119.8 (s, 1C), 119.77 (s, 1C),
113.3 (s, 1C), 105.8 (s, 1C), 70.8 (s, 1C), 60.6 (d, JPC = 6.8 Hz, 1C), 36.3
(d, JPC = 83.0 Hz, 1C), 29.6 (d, JPC = 92.0 Hz, 1C), 16.6 (d, JPC = 5.6 Hz,
1C), 15.5 (d, JPC = 7.9 Hz, 1C), 15.4 (d, JPC = 3.5 Hz, 1C). LC-MS: Rt =
3.28 min. (ESI): m/z = 502 [M + H]+.
Ethyl (3-{[6-(3-Methoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (85). According to the general coupling procedure,
4-chloro-6-(3-methoxyphenyl)-pyrimidine 13h (0.33 g, 1.5 mmol) and
ethyl (3-aminophenyl)propylphosphinate·HCl 48 (0.437 g, 1.65 mmol)
were reacted. The crude product was purified by chromathography in
hexane:EtOAc (9:1). Evaporation of the fractions under reduced
pressure provided a pale-yellow oil (0.18 g, 28%). 31P NMR (CDCl3,
121 MHz): δ 53.2 ppm. 1H NMR (CDCl3, 500 MHz): δ 8.81 (s, 1H),
8.70 (s, 1H), 7.59 (s, 1H), 7.55 (s, 1H), 7.46 (d, JHH = 8.0 Hz, 1H), 7.35
(d, JHH = 8.0 Hz, 1H), 7.3 (t, JHH = 8.0 Hz, 1H), 7.23 (t, JHH = 8.0 Hz,
1H), 7.07 (s, 1H), 6.95 (t, JHH = 8.0 Hz, 2H), 3.9−4.1 (m, 2H), 3.82 (s,
3H), 3.13 (d, JPH = 16.0 Hz, 2H), 1.59−1.63 (m, 4H), 1.24 (t, 7.0 Hz,
3H), 0.97 (t, JHH = 7.0 Hz, 3H). 13C NMR (CDCl3, 75 MHz): δ 162.7 (s,
1C), 161.4 (s, 1C), 159.9 (s, 1C), 158.4 (s, 1C), 139.6 (d, JPC = 2.7 Hz,
1C), 132.9 (d, JPC = 7.3 Hz, 1C), 129.7 (s, 1C), 129.3 (d, JPC = 2.4 Hz,
1C), 124.8 (d, JPC = 5.6 Hz, 1C), 122.4 (d, JPC = 5.3 Hz, 1C), 119.6 (d,
JPC = 2.8 Hz, 1C), 119.2 (s, 1C), 116.1 (s, 1C), 112.2 (s, 1C), 105.7 (s,
162.0 (s, 1C), 161.0 (s, 1C), 158.4 (s, 1C), 157.2 (s, 1C), 139.3 (d, JPC
=
2.6 Hz, 1C), 133.5 (d, JPC = 7.1 Hz, 1C), 131.1 (s, 1C), 130.9 (s, 1C),
129.6 (d, JPC = 2.6 Hz, 1C), 126.9 (s, 1C), 125.7 (d, JPC = 5.5 Hz, 1C),
123.5 (d, JPC = 5.5 Hz, 1C), 121.0 (s, 1C), 120.5 (d, JPC = 3.0 Hz, 1C),
112.6 (s, 1C), 105.3 (s, 1C), 70.3 (s, 1C), 60.7 (d, JPC = 6.7 Hz, 1C), 36.5
(d, JPC = 83.5 Hz, 1C), 29.8 (d, JPC = 92.6 Hz, 1C), 22.7 (s, 1C), 16.8 (d,
JPC = 5.6 Hz, 1C), 15.7 (d, JPC = 5.7 Hz, 1C), 15.5 (d, JPC = 5.6 Hz, 1C),
10.7 (s, 1C). LC-MS: Rt = 3.09 min. (ESI): m/z = 454 [M + H]+.
Ethyl (3-{[6-(2-Isopropoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (82). According to the general coupling procedure,
4-chloro-6-(2-isopropoxyphenyl)-pyrimidine 13e (0.243 g, 0.98 mmol)
and ethyl (3-aminophenyl)propylphosphinate·HCl 48 (0.286 g, 1.08
mmol) were reacted. The crude product was purified by chromathog-
raphy in hexane:EtOAc (9:1). After evaporation of the fractions under
reduced pressure, the product was crystallized from hexane:i-PrOH
(9:1), affording white crystals (0.17 g, 38%). 31P NMR (CDCl3, 121
MHz): δ 52.4 ppm. 1H NMR (CDCl3, 500 MHz): δ 8.77 (s, 1H), 7.43−
7.46 (m, 2H), 7.33−7.37 (m, 2H), 7.27−7.29 (m, 2H), 7.04−7.09 (m,
3H), 6.97 (d, J = 8.0 Hz, 1H), 4.54−4.63 (m, 1H), 3.95−4.07 (m, 2H),
3.13 (d, JPH = 16.5 Hz, 2H), 1.61−1.64 (m, 4H), 1.26 (t, JHH = 6.0 Hz,
9H), 0.97 (t, JHH = 7.5 Hz, 3H). 13C NMR (CDCl3, 75 MHz): δ 162.0 (s,
1C), 101.7 (s, 1C), 60.7 (d, JPC = 6.7 Hz, 1C), 55.3 (s, 1C), 36.4 (d, JPC
=
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82.5 Hz, 1C), 29.8 (d, JPC = 92.5 Hz, 1C), 16.7 (d, JPC = 5.6 Hz, 1C), 15.7
(d, JPC = 6.9 Hz, 1C), 15.5 (d, JPC = 8.9 Hz, 1C). LC-MS: Rt = 3.06 min.
(ESI): m/z = 426 [M + H]+.
and ethyl (3-aminophenyl)propylphosphinate·HCl 48 (0.235 g, 0.89
mmol) were reacted. The crude product was purified by chromathog-
raphy in hexane:EtOAc (9:1). Evaporation of the fractions under
reduced pressure provided a pale-yellow oil (0.08 g, 20%). 31P NMR
(CDCl3, 121 MHz): δ 52.4 ppm. 1H NMR (CDCl3, 500 MHz): δ 8.73
(s, 1H), 8.04 (s, 1H), 7.67 (s, 1H), 7.43−7.52 (m, 4H), 7.26−7.39 (m,
6H), 7.01−7.07 (m, 3H), 5.11 (s, 2H), 4.0 (m, 2H), 3.13 (d, JPH = 16.0
Ethyl (3-{[6-(4-Methoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (86). According to the general coupling procedure,
4-chloro-6-(4-methoxyphenyl)-pyrimidine 13i (0.21 g, 0.95 mmol) and
ethyl (3-aminophenyl)propylphosphinate·HCl 48 (0.278 g, 1.05 mmol)
were reacted. The crude product was purified by chromathography in
hexane:EtOAc (9:1). Evaporation of the fractions under reduced
pressure provided a pale-yellow oil (0.27 g, 67%). 31P NMR (CDCl3,
121 MHz): δ 52.8 ppm. 1H NMR (CDCl3, 500 MHz): δ 8.69 (s, 1H),
8.19 (s, 1H), 7.93 (d, JHH = 9.0 Hz, 2H), 7.48 (s, 1H), 7.34 (d, JHH = 8.0
Hz, 1H), 7.28 (t, JHH = 7.5 Hz, 1H), 7.02 (t, JHH = 8.5 Hz, 2H), 6.94 (d,
JHH = 9.0 Hz, 2H), 4.05 (m, 2H), 3.83 (s, 3H), 3.14 (d, JPH = 16.5 Hz,
2H), 1.59−1.62 (m, 4H), 1.25 (t, JHH = 7.0 Hz, 3H), 0.98 (t, JHH = 7.3
Hz, 2H), 1.59−1.62 (m, 4H), 1.25 (t, JHH = 8.0 Hz, 3H), 0.97 (t, JHH
=
7.5 Hz, 3H). 13C NMR (CDCl3, 75 MHz): δ 163.2 (s, 1C), 161.6 (s,
1C), 159.4 (s, 1C), 158.8 (s, 1C), 139.3 (s, 1C), 139.2 (s, 1C), 139.1 (s,
1C), 136.9 (s, 1C), 133.4 (d, JPC = 7.3 Hz, 1C), 129.9 (s, 1C), 129.7 (s,
1C), 129.7 (s, 1C), 128.7 (s, 2C), 128.2 (s, 1C), 127.7 (s, 2C), 125.5 (d,
JPC = 5.6 Hz, 1C), 122.9 (d, JPC = 5.3 Hz, 1C), 120.2 (d, JPC = 2.9 Hz,
1C), 119.7 (s, 1C), 117.1 (s, 1C), 113.4 (s, 1C), 101.2 (s, 1C), 70.3 (s,
1C), 60.8 (d, JPC = 6.8 Hz, 1C), 36.6 (d, JPC = 83.0 Hz, 1C), 30.4 (d, JPC
=
Hz, 3H). 13C NMR (CDCl3, 75 MHz): δ 163.0 (s, 1C), 161.5 (d, JPC
9.5 Hz, 1C), 158.7 (s, 1C), 139.5 (d, JPC = 2.8 Hz, 1C), 133.3 (d, JPC
=
=
91.8 Hz, 1C), 16.8 (d, JPC = 5.6 Hz, 1C), 15.8 (d, JPC = 2.7 Hz, 1C), 15.6
(d, JPC = 8.6 Hz, 1C). LC-MS: Rt = 3.58 min. (ESI): m/z = 502 [M +
H]+.
7.2 Hz, 1C), 130.0 (s, 1C), 129.6 (d, JPC = 2.5 Hz, 1C), 128.5 (s, 1C),
125.3 (d, JPC = 5.6 Hz, 2C), 122.9 (d, JPC = 5.4 Hz, 2C), 120.1 (d, JPC
=
Ethyl (3-{[6-(2-Ethylphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (90). According to the general coupling procedure,
4-chloro-6-(2-ethylphenyl)pyrimidine 13m (0.053 g, 0.24 mmol) and
ethyl (3-aminophenyl)propylphosphinate·HCl 48 (0.067 g, 0.24 mmol)
were reacted. The crude product was purified by preparative TLC with
two elutions (100% EtOAc then EtOAc:EtOH (8:1)). The pure product
was extracted from the silica with MeOH. After evaporation of the
volatiles under reduced pressure, a white powder was obtained (0.016 g,
16%). 31P NMR (DMSO-d6, 121 MHz): δ 56.7 ppm. 1H NMR (DMSO-
d6, 300 MHz): δ 9.65 (s, 1H), 8.67 (s, 1H), 7.5−7.7 (m, 2H), 7.2−7.45
(m, 5H), 6.94 (d, JHH = 7.2 Hz, 1H), 6.83 (s, 1H), 3.85−4.00 (m, 2H),
3.16 (d, JPH = 16.8 Hz, 2H), 2.73 (q, JHH = 7.2 Hz, 2H), 1.35−1.70 (m,
2.9 Hz, 2C), 114.2 (s, 1C), 99.9 (s, 1C), 60.8 (d, JPC = 6.7 Hz, 1C), 55.5
(s, 1C), 36.6 (d, JPC = 82.9 Hz, 1C), 29.9 (d, JPC = 91.8 Hz, 1C), 16.8 (d,
JPC = 5.6 Hz, 1C), 15.7 (d, JPC = 4.4 Hz, 1C), 15.6 (d, JPC = 6.9 Hz, 1C).
LC-MS: Rt = 2.90 min. (ESI): m/z = 426 [M + H]+.
Ethyl (3-{[6-(2,3-Dimethoxyphenyl)pyrimidin-4-yl]amino}benzyl)-
propylphosphinate (87). According to the general coupling procedure,
4-chloro-6-(2,3-dimethoxyphenyl)-pyrimidine 13j (0.24 g, 0.96 mmol)
and ethyl (3-aminophenyl)propylphosphinate·HCl 48 (0.278 g, 1.05
mmol) were reacted. The crude product was purified by chromathog-
raphy in hexane:EtOAc (9:1). After evaporation of the fractions under
reduced pressure, the product was crystallized from Et2O to give white
crystals (0.16 g, 37%). 31P NMR (CDCl3, 121 MHz): δ 53.9 ppm. 1H
NMR (CDCl3, 500 MHz): δ 8.66 (s, 1H), 8.49 (s, 1H), 7.41 (s, 1H),
7.33 (d, JHH = 8.0 Hz, 1H), 7.27 (dd, JHH = 8.0 Hz, 2H), 7.15 (t, JHH = 7.5
Hz, 1H), 7.03 (t, JHH = 8.0 Hz, 1H), 6.89 (dd, JHH = 9.5 Hz, 2H), 3.95
(m, 2H), 3.77 (s, 3H), 3.62 (s, 3H), 3.04 (d, JPH = 16.5 Hz, 2H), 1.52−
4H), 1.17 (t, JHH = 6.9 Hz, 3H), 1.1 (t, JHH = 7.5 Hz, 3H), 0.92 (t, JHH
=
6.9 Hz, 3H). LC-MS: Rt = 3.05 min. (ESI): m/z = 423 [M + H]+.
Ethyl [3-({6-[2-(Benzyloxy)phenyl]pyrimidin-4-yl}amino)benzyl]-
ethylphosphinate (91). According to the general coupling procedure,
4-[2-(benzyloxy)phenyl]-6-chloropyrimidine 13g (0.593 g, 2.0 mmol)
and ethyl (3-aminophenyl)ethylphosphinate·HCl 47 (0.553 g, 2.1
mmol) were reacted. The crude product was purified by flash
chromathography in 100% EtOAc. After evaporation of the fractions
under reduced pressure, the desired compound was isolated as white
crystals (0.312 g, 32%); mp = 61−62 °C (EtOAc). 31P NMR (DMSO-
d6, 121 MHz): δ 53.9 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 9.6 (s,
1H), 8.69 (s, 1H), 7.84 (d, JHH = 6.9 Hz, 1H), 7.5−7.6 (m, 2H), 7.15−
7.5 (m, 9H), 7.08 (t, JHH = 7.2 Hz, 1H), 6.94 (d, JHH = 7.2 Hz, 1H), 5.26
(s, 2H), 3.85−4.0 (m, 2H), 3.15 (d, JPH = 16.5 Hz, 2H), 1.55−1.7 (m,
2H), 1.17 (t, JHH = 7.2 Hz, 3H), 0.99 (dt, JPH = 17.7 Hz, JHH = 7.7 Hz,
3H). LC-MS: Rt = 3.11 min. (ESI): m/z = 487 [M + H]+.
[3-({6-[2-(Benzyloxy)phenyl]pyrimidin-4-yl}amino)benzyl]-
ethylphosphinic Acid (92). Hydrolysis of the ester 91 (0.136 g, 0.28
mmol) was carried out according to the method described for
compound 61, providing a yellow oil (0.025 g, 19%). 31P NMR
(DMSO-d6, 121 MHz): δ 29.8 ppm. 1H NMR (DMSO-d6, 300 MHz): δ
9.99 (s, 1H), 8.63 (s, 1H), 7.79 (d, JHH = 6.9 Hz, 1H), 7.59 (d, JHH = 7.2
Hz, 1H), 7.2−7.5 (m, 8H), 7.16 (d, JHH = 8.4 Hz, 1H), 7.0−7.1 (m, 2H),
6.85 (d, JHH = 6.9 Hz, 1H), 5.24 (s, 2H), 2.62 (d, JPH = 16.2 Hz, 2H),
1.0−1.15 (m, 2H), 0.88 (dt, JPH = 15.3 Hz, JHH = 6.9 Hz, 3H). LC-MS: Rt
= 0.44 and 2.79 min. (ESI): m/z = 459 [M + H]+.
[3-({6-[2-(Benzyloxy)phenyl]pyrimidin-4-yl}amino)benzyl]-
propylphosphinic Acid (93). Hydrolysis of the ester 84 (0.14 g, 0.28
mmol) was carried out according to the method described for
compound 61, yielding a yellow oil (0.06 g, 45%). It crystallizes upon
standing; mp = 238−240 °C. 31P NMR (DMSO-d6, 121 MHz): DMSO,
δ 32.3 ppm. 1H NMR (DMSO-d6, 300 MHz): δ 10.08 (s, 1H), 8.63 (s,
1H), 7.78 (d, JHH = 7.8 Hz, 1H), 7.61 (d, JHH = 6.9 Hz, 1H), 7.2−7.6 (m,
8H), 7.17 (d, JHH = 8.1 Hz, 1H), 7.0−7.1 (m, 2H), 6.84 (d, JHH = 7.2 Hz,
1H), 5.24 (s, 2H), 2.64 (d, JPH = 15.6 Hz, 2H), 1.3−1.5 (m, 2H), 1.0−1.2
(m, 2H), 0.8 (t, JHH = 6.6 Hz, 3H). LC-MS: Rt = 2.90 min. (ESI): m/z =
473 [M + H]+.
1.59 (m, 4H), 1.45 (t, JHH = 7.0 Hz, 3H), 0.86 (t, JHH = 7.5 Hz, 3H). 13
NMR (CDCl3, 75 MHz): δ 161.4 (s, 1C), 161.0 (s, 1C), 158.3 (s, 1C),
C
153.0 (s, 1C), 147.6 (s, 1C), 139.5 (d, JPC = 2.7 Hz, 1C), 133.1 (d, JPC
7.1 Hz, 1C), 132.4 (s, 1C), 129.3 (d, JPC = 2.5 Hz, 1C), 124.9 (d, JPC
=
=
5.3 Hz, 1C), 124.3 (s, 1C), 122.4 (d, JPC = 5.5 Hz, 1C), 122.1 (s, 1C),
119.6 (d, JPC = 2.8 Hz, 1C), 113.6 (s, 1C), 105.9 (s, 1C), 60.9 (s, 1C),
60.6 (d, JPC = 6.7 Hz, 1C), 55.9 (s, 1C), 36.5 (d, JPC = 83.0 Hz, 1C), 29.6
(d, JPC = 91.9 Hz, 1C), 16.6 (d, JPC = 5.7 Hz, 1C), 15.5 (d, JPC = 5.9 Hz,
1C), 15.3 (d, JPC = 6.1 Hz, 1C). LC-MS: Rt = 2.83 min. (ESI): m/z = 456
[M + H]+.
Ethyl (3-{[6-(4-Fluoro-2-methoxyphenyl)pyrimidin-4-yl]amino}-
benzyl)propylphosphinate (88). According to the general coupling
procedure, 4-chloro-6-(4-fluoro-2-methoxyphenyl)-pyrimidine 13k
(0.12 g, 0.5 mmol) and ethyl (3-aminophenyl)propylphosphinate·HCl
48 (0.146 g, 0.55 mmol) were reacted. The crude product was purified
by chromathography in hexane:EtOAc (9:1). Evaporation of the
fractions under reduced pressure provided a pale-yellow oil (0.05 g,
23%). 31P NMR (CDCl3, 121 MHz): δ 52.6 ppm. 1H NMR (CDCl3, 500
MHz): δ 8.76 (s, 1H), 7.98 (t, JHH = 7.8 Hz, 1H), 7.78 (s, 1H), 7.44 (s,
2H), 7.04−7.35 (m, 2H), 6.81 (d, JHH = 7.5 Hz, 1H), 6.83 (t, JHH = 8.5
Hz, 1H), 6.71 (d, JHH = 8.5 Hz, 1H), 3.9−4.1 (m, 2H), 3.87 (s, 3H), 3.15
(d, JPH = 16.0 Hz, 2H), 1.59−1.64 (m, 4H), 1.3 (t, JHH = 7.0 Hz, 3H), 1.0
(t, JHH = 7.5 Hz, 3H). 13C NMR (CDCl3, 75 MHz): δ 164.7 (d, JFC
=
249.7 Hz, 1C), 160.8 (d, JPC = 8.4 Hz, 1C), 159.1 (d, JPC = 10.0 Hz, 1C),
158.4 (s, 1C), 139.4 (d, JPC = 2.7 Hz, 1C), 133.4 (d, JPC = 7.2 Hz, 1C),
132.4 (d, JPC = 10.3 Hz, 1C), 129.5 (d, JPC = 2.6 Hz, 1C), 125.3 (d, JPC
=
2.6 Hz, 1C), 122.9 (d, JPC = 3.3 Hz, 1C), 122.7 (s, 1C), 122.6 (s, 1C),
119.8 (d, JFC = 3.1 Hz, 1C), 107.8 (d, JFC = 21.2 Hz, 1C), 105.7 (s, 1C),
99.7 (d, JPC = 25.8 Hz, 1C), 60.8 (d, JPC = 6.8 Hz, 1C), 56.1 (s, 1C), 36.6
(d, JPC = 83.6 Hz, 1C), 29.8 (d, JPC = 92.5 Hz, 1C), 16.8 (d, JPC = 5.6 Hz,
1C), 15.7 (d, JPC = 5.6 Hz, 1C), 15.6 (d, JPC = 5.6 Hz, 1C). LC-MS: Rt =
2.89 min. (ESI): m/z = 444 [M + H]+.
Biology. Protocol for CDK9/Cyclin T1 Kinase Assay. CDK9/CycT1
kinase assays were performed in low protein binding 384-well plates
(Corning 3676). Test compounds were diluted in 100% DMSO to 5
Ethyl [3-({6-[3-(Benzyloxy)phenyl]pyrimidin-4-yl}amino)benzyl]-
propylphosphinate (89). According to the general coupling procedure,
4-[3-(benzyloxy)phenyl]-6-chloropyrimidine 13l (0.241 g, 0.81 mmol)
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Journal of Medicinal Chemistry
Article
mM stock concentration, and then further dilutions were made in H2O
or 100% DMSO to desirable concentrations. Each reaction consisted of
5 nM enzyme: CDK9/CyclinT1 (Proqinase catalogue no. 0371-0345-
1), 400 nM TAMRA-Rbtide (synthetic 15-mer peptide derived from
human retinoblastoma tumor suppressor protein labeled with TAMRA
dye, Genecust Europe), 12 μM ATP (= Kmapp, Sigma-Aldrich) and
kinase buffer: 20 mM MOPS pH 7 (Sigma-Aldrich), 1 mM DTT
(Sigma-Aldrich), 10 mM MgCl2 (Sigma-Aldrich), 0.01% Tween 20
(Sigma-Aldrich). For each reaction, 4 or 6 μL containing TAMRA-
Rbtide, ATP, and kianse buffer were combined with 2 μL of diluted
compound in H2O or 0.028 μL of compound in 100% DMSO. The
kinase reaction was started by the addition of 2 μL of diluted enzyme.
The reaction was allowed to run for 1 h at room temperature. The
reaction was stopped by adding 15 μL of IMAP beads (1:400 beads in
progressive (100% buffer A) 1× buffer). After an additional 1 h,
fluorescent polarization (Ex, 550−10 nm; Em, 590−10 nm; Dich, 561
nm) was measured using an Analyst GT (Molecular devices). IC50
curves were fitted with XLfit curve fitting software.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +36-1-4872080. Fax: +36-1-7999990. E-mail:
gnemeth@vichem.hu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project was supported in part by a National Innovation
Office grant (TECH_08_A2-STEMKILL). G.N. thanks the
French Government Grant for financing his stay at ENSCM and
Dr. Leo Leung for his useful suggestions in the manuscript
preparation. Sz. B. thanks the Hungarian Academy of Sciences
for the financial support under the Jan
Scholarship and OTKA PD 109373. We thank Ferenc Baska for
́
os Bolyai Research
Schrodinger predictions.
̈
Protocol for CDK2/Cyclin E and CDK4/Cyclin D1 Kinase Assays.
The percentage of inhibition for CDK2/CycE; CDK4/CycD1 were
determined by our partners under contract work. These CDK/cyclin
protein kinase complexes were assayed using IMAP FAM FP assay
method. Assays were performed in low protein binding 384-well plates
(Corning 3676). Test compounds were diluted in 100% DMSO to 10
mM stock concentration, and then further dilutions were made in 100%
DMSO to desirable concentrations in 384-well polypropylene plate
(Falcon, 3995). Kinase specific FAM labeled peptide substrate was used
in 400 nM. Optimal kinase buffer conditions were determined for each
CDK kinase. ATP concentration was used at the KmATP, app. Kinase
concentration used to yield 50% substrate turnover as determined in
titration experiment. Optimal kinase incubation time was determined
for each kinase: 6 μL of specific FAM-substrate + ATP in reaction buffer
were added to each well. Then 10.8 nL of test compound in 100%
DMSO was added to wells using Pintool. Then 2 μL of reaction buffer
was added to C-wells, and 2 μL of kinase in reaction buffer were added to
wells except C-wells. After kinase incubation period, 15 μL of IMAP
binding solution were added to each well. After IMAP incubation period,
fluorescence polarization (Ex, 485−20 nm; Em, 530−25 nm; Dich, 505
nm) was measured on Analyst GT (Molecular Devices)
Protocol for Viability of MT4 Cell Lines Assay (MTT Assay). The
cytotoxic effect of selected compounds was measured by means of the
colorimetric assay (MTT). Briefly, 2−3 × 104 MT4 cells per well were
cultured in 96-well plates containing RPMI1640 medium supplemented
with 10% FCS and antibiotics (100 IU/mL penicillin (SIGMA), 100 μg/
mL streptomycin (SIGMA)), and two concentrations of the compounds
to be tested (2500 and 1250 nM). After 7 days of incubation (37 °C, 5%
CO2 atmosphere), 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT; 5 mg/mL dissolved in PBS) was added to
each well. After incubation for 4 h, the cell supernatant was removed and
(according our modification) 100 μL of DMSO was added, and the
contents were mixed thoroughly. The absorbance was measured at 540
nm.
ABBREVIATIONS USED
■
ATP, adenosine triphosphate; AZT, azidothymidine; CDK,
cyclin dependent kinase; Ctrl, control; Cyc, cyclin; FDA, U.S.
Food and Drug Administration; HIV, human immunodeficiency
virus; IC50, half-maximal inhibitory concentration; KI, potassium
iodide; Ki, binding affinity; KmATP, dissociation constant of the
kinase and the ATP; Pd/C, palladium on charcoal; RNA,
ribonucleic acid; SAR, structure−activity relationship; TMSCl,
trimethylsilyl chloride
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S
* Supporting Information
Predicted and measured ADME-T properties of selected
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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