Structure-based design and synthesis of antiparasitic pyrrolopyrimidines targeting pteridine reductase 1

Article abstract of DOI:10.1021/jm500483b

The treatment of Human African trypanosomiasis remains a major unmet health need in sub-Saharan Africa. Approaches involving new molecular targets are important; pteridine reductase 1 (PTR1), an enzyme that reduces dihydrobiopterin in Trypanosoma spp., has been identified as a candidate target, and it has been shown previously that substituted pyrrolo[2,3-d]pyrimidines are inhibitors of PTR1 from Trypanosoma brucei (J. Med. Chem. 2010, 53, 221-229). In this study, 61 new pyrrolo[2,3-d]pyrimidines have been prepared, designed with input from new crystal structures of 23 of these compounds complexed with PTR1, and evaluated in screens for enzyme inhibitory activity against PTR1 and in vitro antitrypanosomal activity. Eight compounds were sufficiently active in both screens to take forward to in vivo evaluation. Thus, although evidence for trypanocidal activity in a stage I disease model in mice was obtained, the compounds were too toxic to mice for further development.

Full text of DOI:10.1021/jm500483b

Article
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Structure-Based Design and Synthesis of Antiparasitic
Pyrrolopyrimidines Targeting Pteridine Reductase 1
Abedawn I. Khalaf,† Judith K. Huggan,† Colin J. Suckling,*,† Colin L. Gibson,† Kirsten Stewart,†
Federica Giordani,‡ Michael P. Barrett,*,‡ Pui Ee Wong,‡ Keri L. Barrack,§ and William N. Hunter*,§
†WestCHEM Department of Pure and Applied Chemistry, University of Strathclyde, 295 Cathedral Street, Glasgow G1 1XL, United
Kingdom
‡Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation and Glasgow Polyomics,
College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom
§Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, United
Kingdom
S
* Supporting Information
ABSTRACT: The treatment of Human African trypanoso-
miasis remains a major unmet health need in sub-Saharan
Africa. Approaches involving new molecular targets are
important; pteridine reductase 1 (PTR1), an enzyme that
reduces dihydrobiopterin in Trypanosoma spp., has been
identified as a candidate target, and it has been shown
previously that substituted pyrrolo[2,3-d]pyrimidines are
inhibitors of PTR1 from Trypanosoma brucei (J. Med. Chem.
2010, 53, 221−229). In this study, 61 new pyrrolo[2,3-
d]pyrimidines have been prepared, designed with input from
new crystal structures of 23 of these compounds complexed
with PTR1, and evaluated in screens for enzyme inhibitory
activity against PTR1 and in vitro antitrypanosomal activity. Eight compounds were sufficiently active in both screens to take
forward to in vivo evaluation. Thus, although evidence for trypanocidal activity in a stage I disease model in mice was obtained,
the compounds were too toxic to mice for further development.
HAT and other infectious diseases. Pteridine reductase (PTR1)
has been proposed to be a good target in African trypanosomes.
The enzyme has been shown to be essential using genetic
methods,4 and it has been targeted by inhibitors of several
compound classes.5−8 Among these, pyrrolopyrimidines are
interesting from the perspective of already possessing biological
activity and providing templates for drug discovery; the
pyrimidine ring and its substituents readily key into nucleobase
and cofactor base binding sites in enzymes, and C5, C6, and N7
are suitable for introducing substituents to control selectivity
and physicochemical properties. In the past 2 years alone,
papers have appeared where such a scaffold has been exploited
for protein kinase inhibition,9−13 topoisomerase inhibition and
antibacterial activity,14−16 anti-inflammatory compounds,17
antiparasitic compounds,18 and dipeptidyl peptidase IV
inhibitors.19 In addition, pyrrolopyrimidines bring with them
the advantage of carrying a pharmacophore with structural
similarity to the recognition motif of the parasite’s P2
aminopurine transporter,20 a membrane protein capable of
accumulating its substrates to internal levels that exceed
INTRODUCTION
■
Parasitic disease remains a major global health problem,
especially in tropical and subtropical countries. The availability
of curative medicines is extremely limited and, such as are
available, are of limited effect. Listed among the most neglected
tropical diseases, Human African trypanosomiasis (HAT)
continues to threaten tens of millions of people across rural
Africa.1 However, concerted efforts in surveillance and
intervention have brought the reported incidence to fewer
than 7000 cases in 2011, with estimates of the total number of
infected people being around 20 000.2 The reduction in cases
from an estimated 300 000 at the turn of the century3 has
stimulated a campaign to seek elimination of Trypanosoma
brucei gambiense-related disease by 2030. The zoonotic nature
of Trypanosoma brucei rhodesiense disease makes it more
difficult to plan an elimination strategy.
The limitations of drugs currently used for HAT are several:
there is a requirement for parenteral administration, severe
toxicity is found in some cases, and increasing resistance is
emerging in the parasites themselves.3 This means that new
drugs remain necessary if elimination is to be achieved and the
threat of resurgence is to be removed. It is important to identify
and to exploit new molecular approaches for the treatment of
Received: March 27, 2014
© XXXX American Chemical Society
A
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Figure 1. Organization of the TbPTR1 active site. (A) A surface representation of the TbPTR1 subunit with cofactor and substrate, folate, bound
(PDB 3bmc) showing key residues that create the active site pocket. Potential hydrogen bonds are depicted as magenta dotted lines, and all atoms
are colored according to atom type: O, red; N, blue; S, gold; P, orange; C, yellow (NADP+), cyan (TbPTR1), or black (folate). Phe97 is not labeled
but is shown as thin cyan lines for clarity. (B) The active site with folate removed and key hydrogen-donor or -acceptor groups circled blue or red,
respectively. Phe97 (thin cyan lines) is directly above the nicotinamide in this view. Scaffolds I and II are shown opposite, and possible hydrogen-
donor or -acceptor groups are designated D or A, respectively. Arrows on the schematic also indicate the intended direction of R1, R2, and R3
substitutions into hydrophobic parts of the active site.
a
Scheme 1. 6-Aryl-5-cyano Derivatives
a
(i) Glacial acetic acid/bromine, RT; (ii) boronic acid, Pd(PPh3)4, 2-propanol/H2O, t-butylamine, microwave at 16 °C/40 min.
external concentrations up to a thousand-fold.21 Previously, we
COMPOUND DESIGN
■
reported that a number of heterocyclic compounds including
substituted pyrrolopyrimidines and furopyrimidines are inhib-
itors of PTR1 from Trypanosoma brucei and Leishmania
major.7,22 Here, we use structure-based design on the
pyrrolopyrimidine template, taking into consideration appro-
priate synthetic strategies, to obtain compounds with anti-
trypanosomal activity in vivo.
Crystallographic studies of fused pyrimidines in the active site
of the enzyme from T. brucei, TbPTR1, showed binding at the
folate site, with the pyrimidine ring sandwiched between the
cofactor nicotinamide and a phenylalanine, forming an array of
hydrogen bonds with catalytic residues and the cofactor
phosphate and ribose.7 Two possible binding poses are
observed. One orientation is termed the substrate-like pose
and corresponds to how substrates such as oxidized pterins and
B
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a
Scheme 2. 4-Alkylamino- and 4-Alkoxy Pyrrolopyrimidines
a
(i) Amine, triethylamine, 1,4-dioxane, microwave 1 h/200 °C; (ii) acetonitrile, dimethylaniline, TEBACl, POCl3, 90 °C/1 h; (iii) either alcohol,
sodium metal, heat 30 °C, or amine, triethylamine, 1,4-dioxane, microwave 20 min, 200 °C; (iv) amine, triethylamine, 1,4-dioxane, microwave 1 h, 20
°C; (v) sodium hydroxide solution, reflux overnight; (vi) glacial acetic acid/bromine, RT; (vii) boronic acid, cesium carbonate, [pd(dppf)Cl2], 2-
propanol/H2O, 100 °C overnight. In compound 10, Cl and Br were used in different experiments.
a
Scheme 3
a
(i) Trifluoroacetic anhydride, RT; (ii) DCM, N-iodosuccinimide, reflux 24 h; (iii) sodium hydride, THF, p-toluenesulfonyl chloride; (iv) DMF,
Cu(I)I, phenyl acetylene, tetrakis(triphenylphosphine)palladium(0), triethylamine, RT; (v) saturated methanolic ammonia at 0 °C (vi) DMF,
Cu(I)I, phenylacetylene, tetrakis (triphenylphosphine)palladium(0), triethylamine, RT; (vii) pyrrolidine, TEA, 1,4-dioxane, microwave reactor for 1
h at 200 °C.
pteridines bind. The second orientation is that displayed by the
archetypal antifolate methotrexate (MTX), in which the
pteridine is rotated 180° about the N2−N5 axis in order to
maximize hydrogen-bonding capacity.23 In each case, the
possibility emerged of introducing substituents at positions
4−7 of the pyrrolopyridine template to direct substituents into
hydrophobic pockets near Cys168, Leu209, Pro210, Met213,
and Trp221. The requirement for transport into trypanosomes
C
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a
Scheme 4
a
(i) Ethyl acetate/water, reflux 18 h; (ii) aqueous solution of NaOH/sulfuric acid
and the possibility that the specific transporters found in
trypanosomes that prefer 4-aminopyrimidine substrates might
concentrate the inhibitors advantageously were also consid-
ered;20,21 2,4-diaminopyrimidines were therefore considered to
be important substructures at the outset of this work.
Recognizing that physicochemical properties also play an
important role in the biological activity of substituted
pyrimidines, 4-alkoxy and 4-alkylamino substituents were
investigated. To engage the hydrophobic pockets of PTR1
evident from crystallographic studies, 5-alkyl, 5-aryl, 6-alkyl, and
6-aryl pyrrolopyrimidines together with 5,6-disubstituted
compounds were all studied (Figure 1); the position and
shape of the hydrophobic pockets influenced the selection of
substituents in compounds for synthesis. In principle, N7 could
also support a substituent, but, as will be shown below, the NH
forms an important hydrogen bond with PTR1; consequently,
N7 substituents were not investigated.
coupling of 6-bromopyrrolopyrimidine 14 led to highly
substituted inhibitor 15.
Polysubstituted pyrrolopyrimidines, in general, were found to
be most active as inhibitors of PTR1 and of T. brucei in culture.
One such compound (20) required a targeted synthesis
(Scheme 3). 4-Chloropyrrolopyrimidine 7, protected by
trifluoracetylation at N2 (16), was iodinated with N-
iodosuccinimide to give 17, which was deprotected to give
intermediate 18. Sonogashira coupling followed by nucleophilic
aromatic substitution provided 20, which was substantially
active both in enzyme assays and against T. brucei in culture.
Improved yields in the Songashira coupling with phenyl-
acetylene were obtained when the 7-N-tosyl derivative (22) was
used.
Recognizing the need for a flexible synthesis leading to
pyrrolopyrimidines with 5-hydrophobic substituents, the
Michael addition-based synthesis used previously22 was
extended to include a wide range of aryl and some aralkyl
substitutents in both 4-oxo- and 4-aminopyrimidine series
(Scheme 4). Although in principle triaminopyrimidine 24
might be expected to be more reactive than its 4-oxo relative
(23), it was found that the opposite was the case; reaction rates
were slower and yields were poorer in the preparation of the
2,4-diamino compounds than in the preparation of the 2-
amino-4-oxo compounds. It is likely that the mildly basic
conditions used for the Michael addition step increased the
reactivity of the 2-amino-4-oxo pyrimidine through formation
of the anion. In this way, two groups of 5-arylpyrrolopyr-
imidines, 4-oxo 27a−d and 4-amino 29a−d, were obtained.
This reaction was also applicable to the synthesis of 4-
alkylamino 31b and 4-alkoxypyrrolopyrimidines 31a and 31c. It
SYNTHETIC CHEMISTRY
■
The readily available 4-oxo-5-cyanopyrrolopyrimidine 1 used
previously7,23−25 was brominated at C-6 to obtain 2, and a
variety of aryl and alkyl substituents were introduced by Suzuki
coupling in 10−60% yields (Scheme 1, 3a−c). Similarly, the
corresponding 2,4-diamino compounds 6a−d were obtained
from 2,4-diaminopyrrolopyrimidines 4 and 5. Hydrogenation of
alkene 6b afforded phenylethyl substituted pyrrolopyrimidine
6d.
4-Alkylamino- (8a, 8b, 12d) and 4-alkoxypyrrolopyrimidines
(12a−c) were prepared from the corresponding 4-chloro
derivatives by direct substitution of 4-chloropyrrolopyrimidine
7 with the appropriate amine or alkoxide (Scheme 2). Suzuki
D
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a
Scheme 5
a
(i) AlCl3; (ii) Br2, HOAc; (iii) DMF, 60 °C, 4 d
is notable in passing that the nitroalkyl intermediates 26, 28,
and 30 were found to be trypanocidal, although they are not
active as inhibitors of PTR1 (Tables 1 and 2).
COMPOUND EVALUATION IN VITRO
■
Target compounds and intermediates were first evaluated by a
spectrophotometric enzyme assay that monitored the decrease
in absorbance at 340 nm as NADPH is oxidized (Supporting
Information). Stock solutions at concentration 100 mM in
100% (v/v) DMSO were prepared and screened in duplicate at
10 and 50 μM against 30 μg mL−1 TbPTR1 (0.96 μM). Several
compounds that failed to dissolve adequately at the desired
concentration were rejected, and this left 102 compounds that
were assayed. Inhibition was calculated as a percentage
compared to assessment in the absence of inhibitor with
background NADPH oxidation subtracted from all measure-
ments. Fifty-four compounds displaying at least 60−70%
inhibition of TbPTR1 at 50 μM were further analyzed at
concentrations from 0.025 to 100 μM, and Kiapp values were
determined, assuming reversible competitive inhibition and 1:1
stoichiometry (Table 1). The Ki of MTX was measured
routinely as a standard.26 For consideration for progression to
in vivo models of sleeping sickness in mice, targets optimally of
less than 100 nM Ki against PTR1 (but acceptably less than 400
nM) and an IC50 of less than 500 nM in an in vitro assay of
The developing SAR and crystal structure information
showed that it was necessary to occupy both hydrophobic
pockets at the active site of PTR1 (Figure 1). For this, a new
synthesis was necessary. Three types of pyrimidine substrate,
2,6-diamino-4-oxo 23, 2,4,6-triamino 24, and 2,6-amino-4-
alkylamino 32, were reacted with diaryl bromoketones 33
prepared by bromination of the corresponding arylmethyl
ketones, which were either commercially available or prepared
by Friedel−Crafts acylation of a suitably substituted benzene
derivative. Condensation of the diarylbromoketones afforded
three series of 5,6-diarylpyrrolopyrimidines, 4-oxo 34a−l, 4-
amino 35a−j, and 4-dimethylamino 36a−e, compounds in low
to moderate yields (Scheme 5.) A single example of a 4-
cyclohexylamino analogue (37) and a morpholinoalkyl
analogue (38), designed to provide improved solubility, were
also prepared using the same methods.
E
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Table 1. Assay Results for Pyrrolopyrimidines as Inhibitors of PTR1 and Anti-trypanosomal Activity Against T. b. brucei in
a
Culture and Human HEK Cells
cpd.
no.
TbPTR1
HEK
(IC50 μM)
R1
R2
R3
(Kiapp μM)
T. b. brucei (IC50 μM)
3a
OH
CN
CN
CN
CN
CN
CN
CN
CN
CN
H
3-MeSO2NH−C6H4
3-MeSO2−C6H4
0.350
0.731
0.137
4.9
NT
NT
NT
NT
3b
OH
NT
3c
OH
−CHCH−Ph
NT
4
NH2
NH2
NH2
NH2
NH2
NH2
H
2440
NT
5
Br
3.317
0.230
0.16
0.24
0.35
4.2
62.36
NT
6a
3-CHO−C6H4
6.03
NT
6b
PhCHCH−
72.52
NT
6c
propargyl
170
NT
6d
Ph(CH2)2
17.08 (HMI-9), 8.42 (CMM)
NT
8a
1-pyrrolidinyl
H
NA
NT
8b
1-thiomorpholinyl
H
H
8.6
80.06
NT
12a
12b
12c
12d
13
OMe
CN
CN
CN
CN
CN
CN
CN
H
∼75
>100
>100
8.8
NA
NT
iPrO
H
7.30
NT
cyclopentyloxy
H
1.04
NT
1-thiomorpholinyl
H
14.33
NT
1-pyrrolidinyl
H
Insol
0.8
340
NT
14
4-pyrrolidinyl
Br
139
NT
15
4-pyrrolidinyl
3-CHO−C6H4−
0.2
7.75
NT
20
1-pyrrolidinyl
propargyl
4-Me−C6H4
4-F−C6H4
Ph
H
0.19
1.2
0.65
NT
27a
27b
27c
27d
27e
29a
29b
29c
29d
31a
31b
31c
34a
34b
34c
34d
34e
34f
34g
34h
34i
34j
34k
OH
H
125
NT
OH
H
1.3
25.11
NT
OH
H
1.2
53.34
NT
OH
Ph(CH2)2
Me
H
0.27
7.3
7620
NT
OH
H
NA
NT
NH2
4-Me−C6H4
4-F−C6H4
Ph
H
0.32
0.48
0.4
170
NT
NH2
H
14.47
NT
NH2
H
41.01
NT
NH2
Ph(CH2)2
4-F−C6H4
4-F−C6H4
4-F−C6H4
Ph
H
0.26
>50
0.56
Insol
1.17
1.06
0.50
0.76
0.25
Insol
>2−10
0.230
0.95
>10
Insol
93.52
NT
Me2CHCH2O
H
1.68 (HMI-9), 2.53 (CMM)
12.1 (HMI-9), 11.5 (CMM)
1.13 (HMI-9), 1.42 (CMM)
0.64 (HMI-9), 0.41 (CMM)
18.2 (HMI-9), 10.4 (CMM)
0.74 (HMI-9), 0.61 (CMM)
1.39 (HMI-9), 0.74 (CMM)
2.48 (HMI-9), 1.56 (CMM)
2.66 (HMI-9), 1.03 (CMM)
0.94 (HMI-9), 0.43 (CMM)
7.38 (HMI-9), 3.20 (CMM)
0.40 (HMI-9), 0.14 (CMM)
3.38 (HMI-9), 2.13 (CMM)
NT
N-cyclohexylamino
H
70.77
81.91
NT
i-PrO
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
Ph
Me
Ph
NT
Ph
4-F−C6H4
4-F−C6H4
4-Cl−C6H4
4-F−C6H4
4-Me−C6H4−
4-Br−C6H4−
C6H4‑
>200
160.6
152.4
183.6
124.2
>100
33.18
>200
>200
4-F−C6H4
4-Cl−C6H4
4-OMe−C6H4‑
4-Me−C6H4‑
Ph
Ph(CH2)2
Ph
4-(Me2CHCH2)−C6H4−
4-MeSO2−C6H4
Ph
>100 (HMI-9), >100
(CMM)
34l
OH
3-Cl−C6H4
Ph
4-FC6H4
0.47
0.59
0.24
0.30
Insol
0.58
Insol
0.135
0.58
1.28
0.29
Insol
1.41 (HMI-9), 0.77 (CMM)
2.25 (HMI-9), 0.58 (CMM)
0.32 (HMI-9), 0.08 (CMM)
0.59 (HMI-9), 0.15 (CMM)
3.55 (HMI-9) 2.02 (CMM)
0.27 (HMI-9), 0.083 (CMM)
0.65 (HMI-9), 0.19 (CMM)
0.97 (HMI-9), 0.25 (CMM)
1.97 (HMI-9), 1.39 (CMM)
7.06 (HMI-9), 6.16 (CMM)
0.39 (HMI-9), 0.19 (CMM)
2.77 (HMI-9), 1.31 (CMM)
57.70
62.88
49.19
47.34
53.64
39.14
47.21
39.63
32.54
18.46
34.59
77.10
35a
35b
35c
35d
35e
35f
35g
35h
35i
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NMe2
Ph
Ph
4-F−C6H4
4-F−C6H4
4-Cl−C6H4
4-F−C6H4
4-Me−C6H4−
4-Br−C6H4−
4-(Me2CHCH2)−C6H4−
4-MeSO2−C6H4
4-F−C6H4
4-F−C6H4
4-Cl−C6H4
4-OMe−C6H4‑
4-Me−C6H4‑
Ph
Ph
Ph
35j
3-Cl−C6H4
4-F−C6H4
36a
4−F−C6H4
F
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Table 1. continued
cpd.
no.
TbPTR1
HEK
(IC50 μM)
R1
R2
R3
(Kiapp μM)
T. b. brucei (IC50 μM)
36b
36c
36d
36e
37
NMe2
NMe2
NMe2
NMe2
4-Cl−C6H4
Ph
4-Cl−C6H4
4-F−C6H4
Ph
Insol
Insol
0.29
0.30
0.20
>10
4.96 (HMI-9), 3.08 (CMM)
3.03 (HMI-9), 1.34 (CMM)
10.64 (HMI-9), 6.11 (CMM)
8.55 (HMI-9), 3.43 (CMM)
2.32 (HMI-9), 1.78 (CMM)
38.1 (HMI-9), 20.1 (CMM)
356.4
53.34
57.77
45.84
25.99
NT
Ph
4-OMe−C6H4−
Ph
4-F−C6H4
Ph
N-cyclohexylamino
OH
38
Ph
4-(1-morpholinyl propyl)−C6H4−
diminazene
0.027 (HMI-9), 0.007
(CMM)
methotrexate
3.66 (HMI-9), 0.011 (CMM)
phenylarsine oxide
1.49
a
CMM refers to Creek’s minimal medium, and HMI-9 is a commercially available medium (see Experimental Section).
Table 2. Assay Results for Nitroalkylpyrimidines as Inhibitors of PTR1 and Anti-trypanosomal Activity Against T. b. brucei in
Culture and Human HEK Cells
cpd. no.
R
X
TbPTR1 (Kiapp μM)
T. b. brucei (IC50 μM)
2.038
HEK (IC50 μM)
26a
26b
26c
26d
28a
28b
28c
28d
30a
30b
30c
OH
4-Me−C6H4
4-F−C6H4
H
>100
>100
>100
>100
Insol
∼50
Insol
>100
>100
>100
Insol
73.36
149.5
134.2
NT
OH
1.857
OH
1.016
OH
Ph(CH2)2
4-Me−C6H4
4-F−C6H4
H
N/A
NH2
NH2
NH2
NH2
0.47
32.45
39.11
34.98
NT
0.16
0.13
Ph(CH2)2
4-F−C6H4−
4-F−C6H4−
4-F−C6H4−
8.82
Me2CHCH2O−
N-cyclohexyl amino
i-PrO
13.9 (HMI-9), 10.6 (CMM)
0.54 (HMI-9), 0.80 (CMM)
15.9 (HMI-9), 8.48 (CMM)
NT
32.62
NT
trypanocidal activity were set. Activity data are reported in
Table 1. As noted above, some nitroalkylpyrimidine inter-
mediates (26, 28, 30) also showed activity in the in vitro assay,
and the results are reported in Table 2. Selected significant
compounds were also assayed for toxicity in human HEK cells.
the suggestion that a more flexible hydrophobic 5-substituent
would not give the required activity, as shown by
arylaminomethyl compounds 27d and 29d. The 4-amino series
(29a−c), however, had several compounds with significant
activity in the PTR1 assay. However, the activity in the cellular
assay was disappointing, suggesting that the anticipated
enhanced uptake into trypanosomes was not occurring.
STRUCTURE−ACTIVITY RELATIONSHIPS
■
When 6-hydrophobic substituents were introduced, com-
pounds with greatly improved PTR1 affinity were obtained. In
4-amino, 4-oxo, and 4-alkylamino series (6, 15), compounds
with good inhibitory activity were obtained; however, none of
these compounds was sufficiently active in cellular assays to
merit progression. Indeed, their activity was exceptionally low.
It is possible that the 5-cyano group is sufficiently decreasing
the basicity of these compounds so that they are not substrates
for the transporters. Several compounds with more extended
hydrophobic side chains, notably, phenylethyl, in both the 4-
amino and 4-oxo series also had good inhibitory activity against
PTR1 (6c, 6d, 27d, 29d). One of these (6c) was active enough
in the anti-trypanosomal assay in CMM medium to be
considered for in vivo evaluation. More active compounds
were found, however. Further investigations of substituent
tolerance at C4 showed that alkoxy substitution afforded
insoluble or weakly active compounds (31a, 31c) but that
significant or good enzyme inhibitory activity was obtained with
cyclohexylamino pyrrolopyrimidines (31b, 37). Once again, the
cellular activity was lower than required for progression.
The first variation explored was the substitution of the 4-amino
or 4-oxo group by 4-alkylamino and 4-alkoxy. This was done
using 5-cyanopyrrolopyrimidines (3, 12−15), a class of
compounds that had been found active in previous work, and
two 5-unsubstituted compounds (8a,b), but the general lack of
significant activity together with the crystallographic informa-
tion (see below) implied that a hydrophobic substituent on the
pyrrole ring was necessary for activity.
The cyano vector in the crystal structures suggested that an
appropriate variation was to examine hydrophobically sub-
stituted alkynes with a terminal hydrophobic substituent. The
multistep synthesis leading to 20 was not convenient but did
lead to a compound with good activity at the PTR1 and
acceptable activity against T. brucei in vitro; 20 was taken
forward for further evaluation as described below. The
importance of a significantly sized hydrophobic substituent
was emphasized by the low activity of 5-methylpyrrolopyr-
imidine 27e in the 4-oxo series and 5-cyanopyrrolopyrimidine 4
in the 4-amino series. In the 4-oxo series, 5-aryl substituents
(27) improved the activity in all assays but not sufficiently to
give compounds potent enough for progression. There was also
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Figure 2. Inhibitors overlaid on a van der Waals surface representation of the TbPTR1 active site. Five amino acids that help to cover the ligand-
binding site have been removed for clarity (Phe97, Pro210, Ala212, Met213 and Glu217). NADP+ is shown as yellow sticks. All other atoms are
colored according to element (C, gray; N, blue; O, red; S, yellow; F, pale blue; Br, brown). The orientation is similar to that used in Figure 1.
Figure 3. Difference density omit maps of 23 inhibitors. Difference density (Fo − Fc, where Fo represents the observed and Fc represents the
calculated structure factors) maps were calculated (blue chicken wire) with the molecule removed from the final model and are contoured at 3σ. i
and ii indicate the primary and secondary molecules of 8b when two were simultaneously observed in the active site, whereas A and B indicate
molecules modeled in subunits A or B for 34i. The inhibitors are shown in the same orientation with respect to the active site.
5-Methyl-6-phenyl pyrrolopyrimidine 34b was only modestly
active, showing that more than a 6-aryl substituent was
necessary for useful activity. A significant step forward came
when two aryl substituents were introduced at both C5 and C6,
as shown first by 5,6-diphenylpyrrolopyrimidines 34a and 35a
This led to a clear increase in the anti-trypanosomal assay in
CMM medium and, in the 2,4-diamino case (35a), to a
compound that was at the margin as a further candidate for
progression. A number of such compounds were made, and
several of them displayed activity sufficient for further
progression to in vivo evaluation. Notable, in terms of efficacy,
are 5-phenyl-6-(4-fluorophenyl)pyrrolopyrimidine 35b and 5,6-
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a
Table 3. Pharmacokinetic Properties of Selected PTR1 Inhibitors
TbPTR1
Kiapp μM
T. b. brucei HMI-9
IC50 μM
T. b. brucei CMM
IC50 μM
HEK
Cli
plasma bind
fu
t1/2
clogP
LE PTR1
LE CMM
IC50 μM
mL/min/g
min
20
0.19
0.48
1.17
0.50
0.95
0.47
0.59
0.24
0.65
14.5
5.6
1.7
3.35
1.84
2.75
2.90
3.58
3.26
3.41
3.57
0.4
29b
34a
34c
34i
34l
35a
35b
0.48
0.35
0.36
0.33
0.35
0.37
0.38
0.640
0.738
0.396
1.405
2.247
0.321
0.407
0.614
0.135
0.767
0.583
0.082
1.17
0.17
2.5
0.029
0.017
∼130
0.38
0.35
0.37
0.33
0.37
0.4
>200
33.2
57.7
62.9
49.2
<0.5
5.26
1.90
0.035
0.011
∼100
>500
a
Blank cells imply not tested in relevant assay. Kiapp is the apparent dissociation constant for the enzyme inhibitor complex, before correction for the
inhibition modality-specific influence of substrate concentration relative to Km. As the inhibitors compete for binding with the pterin substrate, Ki can
be calculated according to the equation Kiapp = Ki /(1 + S × Km−1), where S and Km refer to the pterin substrate. Kiapp and IC50 values refer to the
data presented in Tables 1 and 2. Cli is the clearance in vivo. fu is the fraction unbound by plasma. t1/2 is the half-life of the compound. clogP and
ligand efficiency (LE) were obtained using Pipeline Pilot (Accelrys Inc).
di(4-fluorophenyl)pyrrolopyrimidine 35c in the diamino series.
The 4-dimethylamino compounds (36a−e), on the other hand,
were insufficiently active. In the 4-oxo series especially,
solubility prevented good enzyme assays from being obtained
in some cases, but several compounds appropriate for
progression were identified including 34g, 34l, 35f, 35g, and
35j. With the intention of filling the hydrophobic pockets as
completely as possible, a branched alkyl substituent was
introduced (34j, 35h), but this change did not improve
activity. Similarly, the introduction of a strongly electron-
withdrawing group (sulfone) gave only weakly active
compounds with poor solubility (34k, 35i). An attempt to
improve solubility with a flexible, polar ionic substituent (38)
gave a compound with very low activity, and this strategy was
not investigated further.
and van der Waals interactions similar to substrate and MTX
binding poses and retain potency against PTR1.
The compound series that has been developed displayed
good levels of PTR1 inhibition that are based on exploiting
previously recognized molecular features, for example, by
fulfilling the hydrogen-bonding capacity at the catalytic center.
The widely dispersed appearance of the mainly hydrophobic
substituents on the core scaffold (Figure 2) demonstrates the
conformational diversity available to interrogate the different
regions of the active site, and this enhanced affinity for the
target in some cases. In general, the key to improving potency,
as exemplified by 3c, 20, and 35g, has been to incorporate a
sufficiently bulky group capable of additional van der Waals
interactions in the hydrophobic cavities adjacent to the
substrate-binding site.
The 3-formylphenyl substituted compounds (6a and 15) are
worth specific mention. They were designed to adopt the
substrate-like pose but with the appropriate substituents that
might form a covalent attachment to Cys168. This is indeed
observed with stable thioester linkages being formed,
presumably via oxidation of a first formed thioacetal adduct.
The success here indicates that covalent modification might be
exploited in future.
STRUCTURAL BASIS OF PTR1 INHIBITION
■
In previous work, we developed robust protocols to elucidate
high-resolution structures of PTR1.6−8 This allowed us to
acquire new structural data that were combined with modeling
approaches in an iterative process to guide compound design.
Ultimately, this led us to determine isomorphous structures of
23 cocrystal complexes of the PTR1 tetramer (Figures 2 and 3
and Supporting Information). The high-resolution diffraction
data and excellent quality of the electron density maps were
sufficient to identify examples where multiple conformations
and ligand orientations were present, for example, 8b, 29a, and
34i.
COMPOUND EVALUATION IN VITRO
■
Compounds were tested for activity, in vitro, against T. b. brucei
using the Alamar blue assay, which demonstrates cell viability.
Cells were tested in both rich HMI-9 medium and also a
minimal medium, CMM, that does not contain added folate or
biopterin. Results are shown in Tables 1 and 2. When inhibitors
of dihydrofolate reductase (DHFR) are assessed in the latter
medium, they are several hundred-fold more active than when
tested in the rich medium, presumably due to the ability of the
abundant folate to compete for binding sites on the target
enzyme. The fact that for our compounds there was little
difference in potency when using CMM or HMI-9 media would
indicate that they are not exerting their activity through
inhibition of DHFR, a fact consistent with structural
predictions based on modeling within the DHFR-binding site
that suggest regions of steric clash (data not shown).
Superposition of the inhibitors as they are positioned in the
TbPTR1 active site indicates that the core scaffold position and
the hydrogen-bonding interactions of which it is capable are
well-conserved. In addition, most of the inhibitors adopt the
substrate-like pose described earlier. There are three
exceptions: 2,4-diamino-5-phenylethylpyrrolopyrimidine 29d
adopts the MTX-type orientation, 2,4-diamino-5-methyl
analogue 29a displays both the substrate and MTX-type
orientations, and 4-thiomorpholino-substituted compound 12d
displays a distinct binding mode shifted away from the cofactor
and catalytic residues. In all cases, we attribute the change in
orientation to the presence of bulky hydrophobic substituents
that would clash with polar components of the active site. 12d
is unable to mimic interactions that stabilize substrate binding
and therefore is only a modest inhibitor. The other two
compounds (29a and 29d) participate in hydrogen bonding
Compounds were also tested against additional T. b. brucei
strains that are defective in transporter proteins that might be
expected to contribute to their uptake.20,21 The TbAT1
knockout (KO) line has lost the P2 aminopurine transporter,
which is also known to carry melaminophenylarsenicals and
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diamidines,27 whereas the B48 line has additionally lost another
diamidine/melaminophenylarsenical transporter, the HAPT1
carrier (now known to be an aquaglyceroporin designated
AQP2).28,29 Trypanocidal activity on these two transporter
mutants showed hardly any changes as compared with that of
wild-type trypanosomes (data not shown), indicating that our
compounds enter via routes other than, or in addition to, the
P2 transporter and HAPT1. PTR1 inhibitors were also tested
against related leishmania parasites (Leishmania mexicana
M379, data not shown), where activity was moderate and
weaker, indicating differences in its PTR1 from that of
trypanosomes and in its biological context.
showed a relapse of parasitaemia on day 10. The 4-oxo series
was also evaluated with the hope that toxicity would be
reduced. 34h and 34i did not show acute toxicity when
administered at a single dose of 30 mg/kg. However, 34i was
not curative, and toxicity was still observed with 34h, although
parasitaemia was greatly reduced with this compound.
It is evident that there is significant host toxicity in both the
4-oxo and 2,4-diamino series of 5,6-diarylpyrrolopyrimidines
that could not be removed by aryl substituent modification.
This series of compounds is nevertheless trypanotoxic and
inhibits PTR1 in in vitro assays. The detailed information
obtained from the crystallographic studies provides a strong
basis for the design of further series of compounds based on
interactions other than those in the hydrophobic pocket, for
example, the covalent binding described above. Future work
will address whether they actually act through inhibiting this
target in vivo. The compounds demonstrate mammalian
toxicity, possibly through interaction with a CNS receptor,
that must also be addressed in future chemical modifications.
PHARMACOKINETIC AND IN VIVO EVALUATION
■
Progression of compounds into in vivo evaluation was based on
a target property profile that included the following data: (1)
affinity of compound for TbPTR1 preferably <100 nM but
acceptably lower than 400 nM. Several compounds were found
with Ki in the range 120−140 nM, but selection of compounds
for in vivo evaluation also took strongly into account the
activity against T. brucei in culture. The use of a high substrate
concentration to ensure a robust assay implies equally a high
value for Ki in the enzyme assays. (2) Selectivity of compound
with respect to human dihydrofolate reductase, which was
inferred from the relative activity in HDMI and CMM media.
(3) Time required to kill T. brucei in culture desirably within 6
h but acceptably within 48 h; many compounds were found
that killed within 48 h, but none was as rapid as 6 h (kill curves
and the methods used are described in the Supporting
Information). (4) Physicochemical parameters (Table 3):
logP range desirably of 2−3.5 and pKa between 4.5 and 9.2.
For the most active compounds, clogP was in the range 3−3.6
and calculated pKa was typically 4−5 for 4-amino compounds
and 9−9.6 for 4-oxo compounds (Pipeline Pilot, Accelrys Inc.,
was used for both logP and pKa estimation, see the Supporting
Information). (5) Metabolic stability in microsomal assay:
desirably t1/2 > 6 h but acceptable >2 h; t1/2 for the most active
compound (35b) was >8 h. (6) In vitro trypanocidal activity
desirably IC50 < 100 nM but acceptably <800 nM; several
compounds were obtained with IC50 < 500 nM (Table 1).
Compounds for which PK data were obtained are shown in
Table 3. These showed selectivity for killing parasites over a
human cell line (HEK) in culture, again indicative of on-target
activity and at an appropriate level of discrimination to justify
further progression. Ligand efficiencies with respect to both
PTR1 inhibition and in vitro anti-trypanosomal activity were all
good, and the PK properties were acceptable for in vivo
evaluation. LC−MS measurements showed significant accumu-
lation of 34a and 35a into trypanosomes. The PK data showed
good stability and relatively slow clearance, particularly for 35b,
one of the most potent compounds. Four compounds in the 4-
amino series (35b, 35e, 35g, 35j) have been evaluated for
efficacy in vivo. On observation of toxicity in the first round of
experiments, MTD studies with 35b, 35e, and 35g showed
severe toxicity and fatality at 100 mg/kg, some transient clinical
signs at 30 mg/kg, but no clinical signs at 10 mg/kg; therefore,
30 mg/kg was selected as the dose for in vivo efficacy studies.
Infected mice were found to be more sensitive to the toxic
effects of the drugs, but once daily dosing only up to 4 days was
found to be tolerated. In the experiments with 35b, 35e, and
35g, one mouse in each group survived long enough to
demonstrate reduction of parasitaemia from ∼108 to below
detectable limits. The surviving mice receiving 35e and 35g
EXPERIMENTAL SECTION
■
Cell culture. Trypanosomes. Bloodstream form T. b. brucei (strain
Lister 427) was cultured at 37 °C in a humidified 5% CO2
environment in either HMI-9 (Gibco) or Creek’s minimal medium
(CMM)30 supplemented with 10% FBS. Cells were routinely diluted
when reaching the mid-log-phase concentration of 2 × 106 cells/mL
and maintained in culture for no more than 30 passages.
Human Cells. The human embryonic kidney cell line HEK 293T
was cultured in DMEM (Sigma-Aldrich) supplemented with penicillin
(100 units/ml), streptomycin (0.1 mg/mL), L-glutamine (2 mM), and
10% FBS. Cells were grown in a humidified atmosphere at 37 °C and
5% CO2 and split when 80−85% confluent.
Alamar Blue Assay. Against Trypanosomes. Compounds’
potency against trypanosomes was assessed by Alamar blue assay.31
Cells were seeded at a final concentration of 2 × 104 cells/mL into
serial dilutions of the test compound. After 48 h incubation at 37 °C
and 5% CO2, 20 μL of resazurin dye (Sigma-Aldrich) solution at 0.49
mM was added, and cells were incubated for a further 24 h. The
reduction of resazurin was measured with a fluorimeter (FLUOstar
Optima, BMG Labtech) using 544 nm excitation and 590 nm emission
wavelengths. Output was plotted using the IC50 determination
algorithm of the GraphPad Prism 5.0 software. All experiments were
carried out in duplicate and repeated on at least three independent
occasions.
Against HEK Cells. To test compounds’ activity against HEK cells, 3
× 105 cells/mL were seeded in a 96-well plate and allowed to adhere
for 3 h at 37 °C and 5% CO2. An equal volume of doubling serial
dilutions of test compound was then added to the wells, and cells were
incubated for a further 16 h before addition of resazurin (20 μL of a
0.49 mM solution). After 24 h, fluorescence was measured and data
analyzed as described above.
In Vivo Experimental Protocol. Experimental groups of three ICR
mice were infected with 1 × 105 bloodstream T. b. brucei strain 427
obtained from a donor mouse. Twenty-four hours postinfection mice
were injected intraperitoneally with 30 mg/kg daily of test compound
(repeated for four consecutive days), prepared in a 10% v/v DMSO,
40% v/v PEG, and 50% v/v water solution. Parasitaemia was
monitored daily by tail venipuncture. Infected, untreated mice were
used as control. All procedures were carried out by licensed animal
workers and complied with the UK Animals (Scientific Procedures)
Act, 1986, and with the national and University of Glasgow
maintenance and care guidelines.
1
Compound Synthesis. General. H and 13C NMR spectra were
measured on a Bruker DPX 400 MHz spectrometer with chemical
shifts given in ppm (δ values) relative to proton and carbon traces in
solvent. Coupling constants are reported in Hz. IR spectra were
recorded on Shimadzu IRAffinity-1 spectrometer. Elemental analysis
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was carried out on a PerkinElmer 2400 analyzer series 2. Accurate
mass was measured using Thermo Exactive MS and a Thermo U3000
HPLC system. Ionization was carried out by an ESI source (not
heated). Anhydrous solvents were obtained from a Puresolv
purification system, from Innovative Technologies, or purchased as
such from Aldrich. Melting points were recorded on a Reichert hot-
stage microscope and are uncorrected. Chromatography was carried
out using 200−400 mesh silica gels, or using reverse-phase HPLC on a
Waters system using a C18 Luna column.
with methanol, and then silica gel was added. Solvents and excess
reagents were removed under reduced pressure. The residue was
applied to a silica gel column and eluted with methanol/ethyl acetate
(1:9), Rf = 0.2. The required product was obtained as light brown solid
1
(80 mg, 66%), mp > 230 °C. H NMR (DMSO-d6): 10.72 (1H, s),
6.67 (1H, q, J = 1.7 Hz), 5.40 (2H, br), 6.34 (1H, q, J = 1.1 Hz), 3.64
(4H, br), 1.93 (4H, br). IR (KBr): 1605, 1564, 1506, 1457, 1407,
1349, 1195, 1098, 1032, 905, 823, 696 cm.−1 HRESIMS: calculated for
C10H14N5, 204.1244; found, 204.1242.
Declaration of Purity. All final compounds were equal to or more
8b was similarly prepared.
1
than 95% pure by HPLC and H NMR (400 MHz).
N-[5-Cyano-4-(1-pyrrolidinyl)-7H-pyrrolo[2,3-d]pyrimidin-2-
yl]-2,2-dimethylpropanamide (11). N-(4-Chloro-5-cyano-7H-
pyrrolo[2,3-d]pyrimidin-2-yl)-2,2-dimethylpropanamide (350 mg,
1.260 mmol) was suspended in butanol (20 mL), to which was
added pyrrolidine (269 mg, 313 μL, 3 mol equiv, 3.78 mmol). The
reaction mixture was heated at 90 °C in a sealed tube for 48 h. The
reaction mixture was left to cool to room temperature, and the
precipitate was filtered, washed with water, and dried. The required
product (0.290 g) was obtained as brown solid with no distinct
melting point. The filtrate was collected, the organic layer was
separated, and butanol was removed in vacuo and then triturated with
small amount of ethyl acetate and filtered to give an additional amount
of the required material. The total amount obtained was 0.330 g, 84%.
1H NMR (DMSO-d6): 12.52 (1H, br), 9.17 (1H, s), 8.11 (1H, s), 3.78
2-Amino-4-oxo-6-[(E)-2-phenylethenyl]-4,7-dihydro-3H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile (3c). (E)-2-Phenylethe-
nylboronic acid (93 mg, 0.630 mmol), 2-amino-6-bromo-4-oxo-4,7-
dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (80 mg, 0.315
mmol), and cesium carbonate (513 mg, 1.575 mmol) were suspended
in isopropyl alcohol/water (2:1) (6 mL), to which was added (1,1′-
bis(diphenylphosphino)ferrocene)-dichloropalladium(II) [Pd(dppf)-
Cl2] (29 mg, 0.0395 mmol). The reaction mixture was purged with
nitrogen for 20 min and then heated overnight at 100 °C [oil bath
temperature] in a sealed tube. Silica gel was added to the reaction
mixture, and the solvents were removed under reduced pressure. The
residue was partitioned between ethyl acetate and water, the organic
layer was collected, and the solvent was removed. The crude material
was purified by HPLC. Fractions containing the required material were
collected and freeze-dried to give the desired product as a yellow solid
(4H, t, J = 6.5 Hz), 1.98 (4H, t, J = 6.5 Hz), 1.20 (9H, s). IR: 2220,
1706, 1579, 1546, 1425, 1380, 1172, 1105, 807, 788 cm−1 HRESIMS:
calculated for C16H19ON6, 311.1626; found, 311.1628.
1
(20 mg, 23%), mp > 230 °C. H NMR (DMSO-d6): 12.28 (1H, s),
10.73 (1H, s), 7.55 (2H, d, J = 7.4 Hz), 7.42−7.30 (4H, m), 6.99 (1H,
d, J = 16.4 Hz), 6.50 (2H, br). IR (KBr): 2209, 1701, 1609, 1580,
1488, 1419, 1273, 1037, 954, 791, 753, 695 cm.−1 HRESIMS:
calculated for C15H12N5O, 278.1036; found, 278.1039.
2-Amino-4-(1-pyrrolidinyl)-7H-pyrrolo[2,3-d]pyrimidine-5-
carbonitrile (13). N-[5-Cyano-4-(1-pyrrolidinyl)-7H-pyrrolo[2,3-d]-
pyrimidin-2-yl]-2,2-dimethylpropanamide (330 mg, 1.057 mmol) was
suspended in a solution of ethanol (2 mL), sodium hydroxide (85 mg,
2.113 mmol), and water (2 mL). The reaction mixture was heated
under reflux overnight. Solvents were removed under reduced
pressure, and the crude material was suspended in water and ethyl
acetate. The solid material was filtered of to give the required product
(230 mg, 95%) as white solid with no distinct melting point. TLC
3a,b were similarly prepared.
2,4-Diamino-6-bromo-7H-pyrrolo[2,3-d]pyrimidine-5-car-
bonitrile (5). To a suspension of 2,4-diamino-7H-pyrrolo[2,3-
d]pyrimidine-5-carbonitrile (1.087 g, 6.24 mmol) in glacial acetic
acid (70 mL) was added bromine (480 μL, 9.40 mmol, 1.5 equiv). The
reaction mixture was stirred at room temperature for 20 h and then
heated at 60 °C for 14 h. The solvent was removed under reduced
pressure, and the solid material obtained was dried in vacuo for 12 h to
give the required product as a dark brown solid (1.240 g, 79%) with no
1
[ethyl acetate: Rf = 0.5]. H NMR (DMSO-d6): 12.49 (1H, br), 7.99
(1H, s), 6.85 (2H, br), 3.77 (4H, s), 1.99 (4H, s). IR: 2226, 1685,
1633, 1591, 1427, 1207, 1137, 841, 803, 762, 724 cm−1 HRESIMS:
calculated for C11H13N6, 229.1196; found, 229.12.
1
distinct melting point. H NMR (DMSO-d6): 7.49 (2H, br, NH2),
2-Amino-6-bromo-4-(1-pyrrolidinyl)-7H-pyrrolo[2,3-d]-
pyrimidine-5-carbonitrile (14). 2-Amino-4-(1-pyrrolidinyl)-7H-
pyrrolo[2,3-d]pyrimidine-5-carbonitrile (230 mg, 1.008 mmol) was
dissolved in glacial acetic acid (20 mL), to which was added bromine
(78 μL, 242 mg, 1.512 mmol). The reaction mixture was stirred at
room temperature overnight. The solvent was evaporated, and the
crude material was purified by HPLC. The required product was
obtained as yellow solid (90 mg, 29%), mp > 230 °C. 1H NMR
(DMSO-d6): 6.77 (1H, br), 4.74 (2H, br), 3.75 (4H, br). IR (KBr):
2226, 1679, 1633, 1584, 1452, 1391, 1200, 1137 cm.−1 HRESIMS:
calculated for C11H12N6Br, 307.0301; found, 307.0297.
7.00 (2H, br, NH2). IR (KBr): 3332, 3168, 2225, 1662, 1583, 1514,
1430, 1385, 1229, 875, 767 cm.−1
2,4-Diamino-6-[(E)-2-phenylethenyl]-7H-pyrrolo[2,3-d]-
pyrimidine-5-carbonitrile (6b). (E)-2-Phenylethenylboronic acid
(117 mg, 0.790 mmol), 2,4-diamino-6-bromo-7H-pyrrolo[2,3-d]-
pyrimidine-5-carbonitrile (100 mg, 0.395 mmol), and cesium
carbonate (643 mg, 1.975 mmol) were suspended in isopropyl
alcohol/water (2:1) (6 mL), to which was added (1,1′-bis-
(diphenylphosphino)ferrocene)-dichloropalladium(II) [Pd(dppf)Cl2]
(29 mg, 0.0395 mmol). The reaction mixture was purged with
nitrogen for 20 min and then heated overnight at 100 °C [oil bath
temperature] in a sealed tube. Silica gel was added to the reaction
mixture, and the solvents were removed under reduced pressure. The
residue was applied to a silica gel column chromatography and eluted
with [1] n-hexane and [2] ethyl acetate [Rf = 0.1]. Fractions
containing the required material were collected, and the solvent was
removed under reduced pressure to give the desired product as a
yellow solid (65 mg, 60%), mp > 230 °C. 1H NMR (DMSO-d6): 12.11
(1H, s), 7.57 (2H, d, J = 7.Hz), 7.42−7.37 (3H, m), 7.34(1H, t, J = 7.4
Hz), 7.05 (1H, d, J = 16.4 Hz), 6.21 (2H, br), 6.01 (2H, br). IR (KBr):
2209, 1701, 1609, 1580, 1488, 1419, 1273, 1037, 954, 791, 753, 695
cm.−1 HRESIMS: calculated for C15H13N6, 277.1196; found, 277.1199.
6a and 6c−e were similarly prepared.
2-Amino-6-(3-formylphenyl)-4-(1-pyrrolidinyl)-7H-pyrrolo-
[2,3-d]pyrimidine-5-carbonitrile (15). 3-(4,4,5,5-Tetramethyl-
1,3,2-dioxaborolan-2-yl)benzaldehyde (76 mg, 0.326 mmol), 2-
amino-6-bromo-4-(1-pyrrolidinyl)-7H-pyrrolo[2,3-d]pyrimidine-5-car-
bonitrile (50 mg, 0.163 mmol), and cesium carbonate (266 mg, 0.815
mmol) were suspended in isopropyl alcohol/water (2:1) (6 mL), to
which was added (1,1′-bis(diphenylphosphino)ferrocene)-
dichloropalladium(II) [Pd(dppf)Cl2] (12 mg, 0.0163 mmol). The
reaction mixture was purged with nitrogen for 20 min then heated
overnight at 100 °C [oil bath temperature] in a sealed tube. Solvents
were removed under reduced pressure, and the residue was purified by
HPLC. The freeze-dried material was obtained as a white solid (10 mg,
1
19%) with no distinct melting point. H NMR (DMSO-d6): 13.06
4-(1-Pyrrolidinyl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine (8a).
A mixture of 4-chloro-7H-pyrrolo[2,3-d]pyrimidin-2-amine (100 mg,
0.593 mmol), pyrolidine (44 mg, 0.622 mmol, 102 μL, d 0.860), and
triethylamine (63 mg, d 0.726, 87 μL, 0.720 mmol) were dissolved in
1,4-dioxane (2 mL, dry). The reaction mixture was heated in a
microwave reactor for 1 h at 200 °C. The reaction mixture was diluted
(1H, br), 10.11 (1H, s), 8.34 (1H, t, J = 1.5 Hz), 8.15 (1H, t, J = 1.2
Hz), 8.07 (1H, t, J = 1.2 Hz), 7.83 (1H, d, J = 7.7 Hz), 6.84 (2H, br),
3.84 (4H, s), 2.02 (4H, s). IR (KBr): 2221, 1659, 1555, 1463, 1388,
1110, 1139, 892, 835, 802, 763, 721 cm.−1 HRESIMS: calculated for
C18H17N6O, 333.1458; found, 333.1460.
K
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N-(4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-2,2,2-trifluor-
oacetamide (16). 4-Chloro-7H-pyrrolo[2,3-d]pyrimidin-2-amine
(1.20 g, 7.118 mmol) was dissolved in pyridine (6 mL, dry), to
which was added trifluoroacetic anhydride (TFAA) (1.3 mL, 9.35
mmol) dropwise within 5 min, and the mixture was stirred for 3 h at
room temperature. The solvent was removed under reduced pressure
to yield an amber solid, which was coevaporated twice with water (5
mL). The resulting material was filtered, washed with cold water, and
then dried to give the required product as light brown solid (0.660 g,
(saturated) solution and ethyl acetate and extracted. The organic layers
were combined, dried (Na2SO4), and filtered, and the solvent was
removed under reduced pressure. The required material was obtained
after recrystallization from ethyl acetate/n-hexane (0.330 g, 61%), mp
1
215−218 °C. H NMR (DMSO-d6): 12.64 (1H, s), 8.32 (2H, d, J =
8.3 Hz), 8.19 (1H, s), 7.47 (2H, d, J = 8.3 Hz), 2.38(3H, s). IR (KBr):
1753, 1595, 1569, 1451, 1375, 1218, 1174, 1141, 1087, 916, 813 cm.−1
HRESIMS: calculated for C15H10ClF3IN4O3S, 544.9153; found,
544.9155.
1
35%), mp 227−229 °C (dec). H NMR (DMSO-d6): 12.65 (1H, s),
N-[4-Chloro-7-[(4-methylphenyl)sulfonyl]-5-(phenylethyn-
yl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]-2,2,2-trifluoroacetamide
(22). N-{4-Chloro-5-iodo-7-[(4-methylphenyl)sulfonyl]-7H-pyrrolo-
[2,3-d]pyrimidin-2-yl}-2,2,2-trifluoroacetamide (300 mg, 0.551
mmol) was dissolved in DMF (10 mL, dry), to which were added
Cu(I)I (10.6 mg, 0.055 mmol, 10% molar equivalents), phenyl-
acetylene (68 mg, 0.661 mmol, 73 μL, 1.2 mol equiv), tetrakis-
(triphenylphosphine)palladium(0) (64 mg, 0.055 mmol, 10% molar
equivalents), and triethylamine (112 mg, 1.102 mmol, 2 mol equiv) at
room temperature with stirring under nitrogen. The reaction mixture
was left stirring at room temperature overnight. Brine was added to the
reaction mixture, and the precipitated brown solid was filtered, washed
with water, and dried to give the desired product as brown solid
(0.2070 g, 72%), mp > 230 °C. 1H NMR (DMSO-d6): 8.10 (2H, d, J =
8.4 Hz), 7.87 (1H, s), 7.55−7.43 (6H, m), 7.34 (2H, s), 2.40 (3H, s).
IR (KBr): 1631, 1602, 1541, 1487, 1378, 1178, 1112, 1013, 786, 755
cm.−1 HRESIMS: calculated for C7H7O2S, 363.0273; found, 363.0275.
2-Amino-5-phenyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-
4-one (27c).32 To an aqueous solution of NaOH (0.330 g, 8.25
mmol) in 5 mL of water was added 2,6-diamino-5-(2-nitro-1-
phenylethyl)-4(3H)-pyrimidinone (0.398 g, 1.53 mmol) at room
temperature. The mixture was stirred for 2 h and then was slowly
added to an aqueous solution of 1.37 g (14 mmol) of sulfuric acid
(98%) in 5 mL of water at 0 °C [the sequence of the addition is
important]. The resulting mixture was stirred at 0 °C for 1 h and at
room temperature overnight. The solid material was filtered, washed
with water, and dried. The crude material was purified by HPLC, and
fractions containing the required product were collected and freeze-
dried to give lilac colored solid (50 mg, 15%), mp > 230 °C. 1H NMR
(DMSO-d6): 11.26 (1H, s), 10.43 (1H, s), 7.95 (2H, dd, J = 1.24 and
8.4 Hz), 7.31 (2H, t, J = 7.52 Hz), 7.16 (1H, t, 7.4 Hz), 7.03 (1H, d, J
= 2.4 Hz), 6.25 (2H, br). IR (KBr): 1719, 1684, 1657, 1210, 1181,
1146, 763, 723, 705 cm.−1 HRESIMS: calculated for C12H11ON4,
227.0927; found, 227.0925.
12.20 (1H, s), 7.66 (1H, dd, J = 3.5 and 1.5 Hz), 6.61 (1H, q, J = 1.7
Hz). IR (KBr): 1732, 1582, 1425, 1338, 1287, 1203, 1159, 925, 887,
823, 772, 737 cm.−1 HRESIMS: calculated for C8H5ON435ClF3,
265.0098; found, 265.0098.
N-(4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-2,2,2-
trifluoroacetamide (17). To a suspension of 4-chloro-7H-pyrrolo-
[2,3-d]pyrimidin-2-amine (0.660 g, 2.494 mmol) in DCM (20 mL,
dry) was added N-iodosuccinimide (0.971 g, 4.315 mmol), and the
reaction mixture was heated under reflux for 12 h. The suspension was
filtered, and the solid was washed with hot water. The product was
obtained as gray solid (0.840 g, 86%), mp > 230 °C. 1H NMR
(DMSO-d6): 13.00 (1H, s), 12.27 (1H, s), 7.89 (1H, d, J = 2.4 Hz). IR
(KBr): 1733, 1576, 1527, 1452, 1422, 1334, 1264, 1186, 1166, 965,
924, 767 cm.−1 HRESIMS: calculated for C8H4ON435ClF3I, 390.9065;
found, 390.9064.
4-Chloro-5-(phenylethynyl)-7H-pyrrolo[2,3-d]pyrimidin-2-
ylamine (19). 4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-2-amine
(300 mg, 1.02 mmol), phenyl acetylene (125 mg, 1.22 mmol, 112 μL,
1.2 mol equiv), Cu(I)I (10 mg), tetrakis (32 mg), and TEA (206 mg,
2.04 mmol, 287 μL, 2 mol equiv) were dissolved in DMF (10 mL,
dry). The reaction mixture was stirred at room temperature overnight.
Brine was added to the reaction mixture (the reaction was slightly
exothermic). The brown solid precipitated was filtered, washed with
water, and dried. This was then triturated with boiling methanol and
filtered hot to give (0.2350 g, 86%) as brown solid. The filtrate was
concentrated and applied to a silica gel column and eluted with ethyl
acetate. The pure material was obtained as pale yellow solid (31 mg,
11%), mp > 230 °C. 1H NMR (DMSO-d6): 11.87, 7.54 (1H, d, J = 2.3
Hz), 7.49−7.47 (2H, m), 7.41−7.36 (3H, m), 6.68 (2H, s). IR (KBr):
2215, 1633, 1560, 1488, 1448, 1404, 1260, 1020, 926, 819, 787, 752,
689 cm.−1 HRESIMS: calculated for C14H10ClN4, 269.0589; found,
269.0592.
5-(Phenylethynyl)-4-(1-pyrrolidinyl)-7H-pyrrolo[2,3-d]-
pyrimidin-2-amine (20). 4-Chloro-5-(phenylethynyl)-7H-pyrrolo-
[2,3-d]pyrimidin-2-ylamine (100 mg, 0.372 mmol), pyrolidine (100
μL, 86 mg, 1.21 mmol), and TEA (100 μL, 72.6 mg, 0.719 mmol)
were added to 1,4-dioxane (4 mL, dry). The reaction mixture was
heated in a microwave reactor for 1 h at 200 °C. The reaction mixture
was diluted with methanol, and then silica gel was added. Solvents and
excess reagents were removed under reduced pressure. The residue
was applied to a silica gel column and eluted with methanol/ethyl
acetate (1:1) containing 1% TEA. The crude material obtained was
further purified by HPLC to give the required product as light brown
solid after freeze-drying (10 mg, 9%), mp > 230 °C. 1H NMR
(DMSO-d6): 12.20 (1H, br), 7.52−7.38 (6H, m), 7.17 (2H, br), 3.93
(4H, br), 2.02 (4H, br). IR (KBr): 2211, 1678, 1635, 1201, 1181,
1136, 884, 836, 800, 758, 722, 688 cm.−1 HRESIMS: calculated for
C18H18N5, 304.1557; found, 304.1561.
N-{4-Chloro-5-iodo-7-[(4-methylphenyl)sulfonyl]-7H-
pyrrolo[2,3-d]pyrimidin-2-yl}-2,2,2-trifluoroacetamide (21). So-
dium hydride (48 mg, 1.20 mmol, 60% in oil, 1.2 mol equiv) was
added to THF (20 mL, dry) under nitrogen. To this was added the
starting material (0.3905 g, 1.00 mmol) while the reaction mixture was
cooled to 0 °C with stirring. The reaction mixture was left stirring for a
further 15 min after completion of the addition. p-Toluenesulfonyl
chloride (229 mg, 1.2 mmol, 1.2 mol equivalents) was added to the
reaction mixture portionwise while keeping the temperature between 5
and 10 °C. After the addition had ended, the mixture was allowed to
come to 20 °C, and the stirring was continued at this temperature for a
further 1 h. The reaction mixture was extracted with aq. NaHCO3
27a,b,d were similarly prepared.
5-(2-Nitro-1-phenylethyl)-2,4,6-pyrimidinetriamine (28c).
2,4,6-Pyrimidinetriamine (0.575 g, 4.60 mmol) and [(E)-2-
nitroethenyl]benzene (0.783 g, 5.25 mmol) were suspended in a
mixture of water (20 mL) and ethyl acetate (20 mL) at room
temperature. The resulting mixture was left stirring at room
temperature for 18 h. The reaction mixture was extracted with ethyl
acetate and dried (Na2SO4). The crude material obtained was purified
by column chromatography using silica gel, methanol/ethyl acetate
[1:9, Rf = 0.5], to give the required product as yellow solid (0.500 g,
1
40%), mp 110−113 °C (transparent). H NMR (DMSO-d6): 7.36−
7.22 (5H, m), 5.48−5.41 (7H, m, containing 3 × NH2, exchangeable),
5.18−5.04 (2H, m). IR (KBr): 1619, 1567, 1442, 1377, 1254, 1032,
802, 743, 701 cm.−1 HRESIMS: calculated for C12H15O2N6, 275.1251;
found, 275.1244.
28a,b,d were similarly prepared.
5-Phenyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (29c).32
To a solution of NaOH (0.330 g, 8.25 mmol) in water (5 mL) was
added 5-(2-nitro-1-phenylethyl)-2,4,6-pyrimidinetriamine (0.441 g,
1.53 mmol) at room temperature. The mixture was stirred for 2 h
and then was slowly added to a solution of sulfuric acid (98%, 1.37 g,
14 mmol) in water (5 mL) at 0 °C [the sequence of the addition is
important]. The resulting mixture was stirred at 0 °C for 1 h and at
room temperature overnight. The solid material was filtered, washed
with water, and dried to give the crude product as brown solid (285
mg). The crude material was purified by HPLC, and fractions
containing the required product were collected and freeze-dried to give
L
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1
pale yellow solid (50 mg, 29%), mp 200−203 °C. H NMR (DMSO-
d6): 11.91 (1H, s), 7.48−7.34 (5H, m), 7.20 (4H, br), 7.08 (1H, d, J =
1.8 Hz). IR (KBr): 1694, 1651, 1543, 1453, 1390, 1207, 1131, 826,
800, 759, 725 cm.−1 HRESIMS: calculated for C12H12N5, 226.1087;
found, 226.1082.
2-Bromo-1-(4-fluorophenyl)-2-phenylethanone (33c). 1-(4-
Fluorophenyl)-2-phenylethanone (5.46 g, 25.49 mmol) was dissolved
in chloroform (58 mL), to which was added a hydrobromic acid
solution 30% in acetic acid (0.140 mL, 1 mol equiv) at room
temperature with stirring. Bromine (1.32 mL) was dissolved in
chloroform (5 mL) and added to the reaction mixture dropwise with
stirring. At the end of the reaction, a slight bromine coloration should
remain. Aqueous sodium sulfite (10%) solution was added, and the
reaction mixture was then extracted. The organic layer was collected
and washed with a saturated aqueous solution of sodium hydrogen
carbonate followed by brine, the organic layer was dried (Na2SO4) and
filtered, and the solvent was removed under reduced pressure to give
the required product (7.20 g, 96%) as a reddish-brown oil that
solidified on standing, mp 45−46 °C (Lit. mp 46 °C).33 1H NMR
(CDCl3): 8.07 (2H, dd, J = 5.4 Hz, 9.0 Hz), 7.55−7.53 (2H, m),
7.43−7.34 (3H, m), 7.17 (2H, dd, J = 8. 5 Hz, 8.8 Hz), 6.34 (1H, s).
IR: 729, 771, 797, 827, 852, 991, 1005, 1103, 1155, 1186, 1213, 1234,
1271, 1302, 1408, 1454, 1495, 1504, 1593, 1688 cm.−1 HRESIMS:
calculated for C14H1179BrFO, 292.9972; found, 292.9974.
2-Bromo-1,4-diphenyl-1-butanone (33a), 2-bromo-1,2-bis(4-
fluorophenyl)ethanone (33d), 2-bromo-1,2-bis(4-chlorophenyl)-
ethanone (33e), 2-bromo-1-(4-fluorophenyl)-2-(4-methoxyphenyl)-
ethanone (33f), 2-bromo-1,2-bis(4-methylphenyl)ethanone (33g), 2-
bromo-1-(4-bromophenyl)-2-phenylethanone (33h), 2-bromo-1-(4-
isobutylphenyl)-2-phenylethanone (33j), 2-bromo-1-[4-
(methylsulfonyl)phenyl]-2-phenylethanone (33k), 2-bromo-2-(3-
chlorophenyl)-1-(4-fluorophenyl)ethanone (33l), and 2-bromo-1-{4-
[3-(4-morpholinyl)propyl]phenyl}-2-phenylethanone (33m) were
similarly prepared.
29a,b,d were similarly prepared.
5-[1-(4-Fluorophenyl)-2-nitroethyl]-6-isopropyloxy-2,4-pyri-
midinediamine (30c). 6-Isopropyloxy-2,4-pyrimidinediamine (250
mg, 1.486 mmol) and 1-fluoro-4-[(E)-2-nitroethenyl]benzene (248
mg, 1.486 mmol) were dissolved in ethyl acetate (20 mL). The
reaction mixture was placed in a sealed tube and heated at 50 °C for 4
days. Solvent was removed under reduced pressure, and the crude
material was applied to a silica gel column chromatography. Elution
with ethyl acetate/n-hexane 1:1 (Rf = 0.1) gave the required product
1
(100 mg, 20%) as white solid, mp 148−150 °C. H NMR (DMSO-
d6): 7.37 (2H, dd, J = 5.5 Hz, J = 8.7 Hz), 7.12 (2H, t, J = 8.9 Hz), 6.14
(2H, s), 5.78 (2H, s), 5.25−5.13 (3H, m), 4.82 (1H, t, J = 8.0 Hz),
1.25 (3H, d, J = 6.2 Hz), 1.08 (3H, d, J = 6.2 Hz). IR (KBr): 798, 818,
1005, 1109, 1140, 1198, 1231, 1375, 1422, 1445, 1508, 1539, 1566,
1603, 1653 cm−1. HRESIMS: calculated for C15H19FN5O3, 336.1466;
found, 336.1469.
5-(4-Fluorophenyl)-4-isopropyloxy-7H-pyrrolo[2,3-d]-
pyrimidin-2-amine (31c). 5-[1-(4-Fluorophenyl)-2-nitroethyl]-6-
isopropyloxy-2,4-pyrimidinediamine (65 mg, 0.194 mmol) was
dissolved in a solution of NaOH (215 mg, 5.375 mmol) in water (5
mL). The reaction mixture was left stirring at room temperature for 2
h (with occasional gentle heating to help the starting material to go
into solution). This solution was added dropwise to a cooled solution
of sulfuric acid (0.70 g) in water (5 mL) at 0 °C with stirring, after
which time the reaction mixture was left stirring at room temperature
overnight. The white solid material formed was filtered, washed with
water, and dried. HPLC purification of this material afforded the
required product (10 mg, 18%) as white solid after freeze-drying with
no distinct melting point. The starting material was also recovered (10
2-Amino-5,6-diphenyl-3,7-dihydro-4H-pyrrolo[2,3-d]-
pyrimidin-4-one (34a). 2,6-Diamino-4(3H)-pyrimidinone (0.290 g,
2.00 mmol) and desyl bromide (0.550 g, 2.00 mmol) were dissolved in
DMF (2 mL, dry). The reaction mixture was heated at 60 °C for 4
days. The solvent was removed in vacuo, and the residue was applied
to a silica gel column and eluted with 1:9 methanol/ethyl acetate. The
product was obtained as yellow solid after trituration with hot
methanol (230 mg, 38%). 1H NMR (DMSO-d6): 11.44 (1H, s), 10.27
(1H, s), 7.31−7.18 (10H, m), 6.13 (2H, s). IR: 698, 724, 758, 783,
835, 880, 1157, 1225, 1377, 1441, 1506, 1516, 1543, 1599, 1634 cm.−1
HRESIMS: calculated for C18H15N4O, 303.1240; found, 303.1242.
2-Amino-6-(4-fluorophenyl)-5-phenyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one (34c). 2-Bromo-1-(4-fluorophen-
yl)-2-phenylethanone (2.324 g, 7.929 mmol) and 2,6-diamino-
4(3H)-pyrimidinone (1.00 g, 7.929 mmol) were dissolved in DMF
(4 mL, dry). The reaction mixture was heated at 60 °C for 4 days with
stirring under nitrogen. DMF was removed in vacuo, and the residue
was applied to a silica gel column chromatography and eluted with
methanol/ethyl acetate (1:9; Rf = 0.5). The product was obtained as
an orange solid (0.880 g, 35%), mp > 230 °C. Some of this material
1
mg, 15%) as pale yellow solid. H NMR (DMSO-d6): 11.50 (1H, s),
7.68 (2H, dd, J = 5.6 Hz, J = 8.9 Hz), 7.20 (2H, t, J = 8.9 Hz), 7.11
(1H, d, J = 2.3 Hz), 6.58 (2H, br), 5.47 (1H, septet, J = 6.2 Hz), 1.33
(6H, d, J = 6.2 Hz). IR (KBr): 1720, 1684, 1658, 1213, 1177, 1141,
833, 723, 707, 686 cm.−1 HRESIMS: calculated for C15H16FN4O,
287.1303; found, 287.1307.
31a,b were similarly prepared.
Leading to 34c, 35b, and 36c. 1-(4-Fluorophenyl)-2-phenyl-
ethanone.33 Aluminum chloride (16.0 g, 0.119 mmol, 1.2 mol equiv)
was added to fluorobenzene (50 mL, 51.2 g, 0.533 mmol, 5.0 mol
equiv) with stirring and cooling with ice water under nitrogen.
Phenacyl chloride (13.8 mL, 16.13 g, 0.104 mmol, 1.05 mol equiv) was
added dropwise while keeping the temperature below 20 °C. The
reaction mixture was stirred for a further 15 min, and then it was
heated at 50 °C for 5 h, after which time the reaction mixture was left
stirring at room temperature for 9 h. Hydrolysis was carried out by
diluting with dichloromethane, pouring the reaction mixture onto
crushed ice (50 g), and extracting the resulting suspension with HCl
(2M, 30 mL). The organic phase was then cautiously washed with a
saturated aqueous solution of sodium hydrogen carbonate and brine.
The organic layer was dried (Na2SO4) and filtered, and the solvent was
removed under reduced pressure to give solid material, which was
washed with n-hexane. The desired material (21.00 g, 94%) was
obtained as a pale yellow solid, mp 83−85 °C (Lit. mp 82 °C),33 Rf =
0.5 (1:6 ethyl acetate/n-hexane). 1H NMR (DMSO-d6): 8.16 (2H, dd,
J = 6.0 Hz, J = 8.3 Hz), 7.39−7.24 (7H, m), 4.39 (2H, s). IR: 710, 722,
744, 792, 830, 860, 990, 1093, 1147, 1191, 1233, 1333, 1413, 1502,
1593, 1677 cm.−1 HRESIMS: calculated for C14H12FO, 215.0867;
found, 215.0870.
1
was further purified by HPLC. H NMR (DMSO-d6): 11.48 (1H, s),
10.30 (1H, s), 7.29−7.19 (7H, m), 7.10 (2H, t, J = 8.9 Hz), 6.16 (2H,
br). IR: 722, 758, 784, 811, 835, 879, 1155, 1225, 1377, 1441, 1506,
1516, 1543, 1600, 1633 cm.−1 HRESIMS: calculated for C18H14ON4,
321.1146; found, 321.1145.
34b−l were similarly prepared.
5,6-Diphenyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine
(35a). 2,4,6-Pyrimidinetriamine (0.250 g, 2.00 mmol) and desyl
bromide (0.550 g, 2.00 mmol) were dissolved in DMF (2 mL, dry).
The reaction mixture was heated at 60 °C for 4 days. The solvent was
removed in vacuo, and the residue was applied to a silica gel column
chromatography and eluted with 1:9 methanol/ethyl acetate, Rf = 0.4.
The product was obtained as yellow solid (68 mg, 11%). Some of this
1
material was further purified by HPLC, mp 130−133 °C. H NMR
1,2-Bis(4-fluorophenyl)ethanone, 1,2-bis(4-chlorophenyl)ethanone,
1-(4-fluorophenyl)-2-(4-methoxyphenyl)ethanone, 1,2-bis(4-
methylphenyl)ethanone, 1-(4-bromophenyl)-2-phenylethanone, 1-(4-
isobutylphenyl)-2-phenylethanone, 1-[4-(methylsulfonyl)phenyl]-2-
phenylethanone, and 2-(3-chlorophenyl)-1-(4-fluorophenyl)ethanone,
and 1-[4-(3-chloropropyl)phenyl]-2-phenylethanone were similarly
prepared.
(DMSO-d6): 12.26 (1H, s), 7.48−7.25 (12H, m), 6.67 (2H, br). IR:
1647, 1443, 1389, 1194, 1136, 1076, 1017, 972, 918, 835, 810, 766,
694 cm−1 HRESIMS: calculated for C18H16N5, 302.1400; found,
302.1396.
6-(4-Fluorophenyl)-5-phenyl-7H-pyrrolo[2,3-d]pyrimidine-
2,4-diamine (35b). 2-Bromo-1-(4-fluorophenyl)-2-phenylethanone
(2.324 g, 7.929 mmol) and 2,4,6-pyrimidinetriamine (1.000 g, 7.991
M
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Journal of Medicinal Chemistry
Article
mmol) were dissolved in DMF (4 mL, dry). The reaction mixture was
heated at 60 °C for 4 days with stirring under nitrogen. DMF was
removed in vacuo, and the residue was applied to a silica gel column
chromatography and eluted with methanol/ethyl acetate (1:9; Rf =
0.4). The product was obtained as yellow solid (0.270 g, 11%), mp
125−130 °C. Some of this material was further purified by HPLC. IR:
721, 758, 783, 810, 835, 879, 1157, 1225, 1377, 1441, 1506, 1516,
1543, 1599 cm.−1 1H NMR (DMSO-d6): 12.32 (1H, s), 7.48−7.26
(9H, m), 7.18 (2H, t, J = 8.9 Hz), 6.79 (2H, br). HRESIMS: calculated
for C18H15N5F, 320.1306; found, 320.1307.
9.50 (1H, br), 7.29−7.08 (9H, m), 6.11 (2H, br), 3.10 (4H, m), 2.59
(2H, m), 1.94 (2H, m). IR: 696, 719, 765, 796, 835, 1132, 1194, 1433,
1495, 1657 cm−1. HRESIMS: calculated for C25H28N5O2, 430.2238;
found, 430.2242.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic methods and characterization of analogues of
compounds described herein; protein preparation and
purification; spectrophotometric assay methods; and ligand
cocrystallization and structure determination. This material is
available free of charge via the Internet at http://pubs.acs.org.
35c−j were similarly prepared.
N4,N4-Dimethyl-5,6-diphenyl-7H-pyrrolo[2,3-d]pyrimidine-
2,4-diamine (36d). N4,N4-Dimethyl-2,4,6-pyrimidinetriamine (0.500
g, 3.26 mmol) and 2-bromo-1,2-diphenylethanone [desyl bromide]
(0.898 g, 3.26 mmol) were dissolved in DMF (4 mL, dry), to which
were added potassium iodide (0.541 g, 3.26 mmol) and cesium
carbonate (1.062 g, 3.26 mmol). The reaction mixture was heated at
60 °C for 24 h. DMF was removed in vacuo, and the residue was
dissolved in ethyl acetate and methanol, to which was added silica gel,
and the solvents were removed under reduced pressure. The residue
was applied to a silica gel column chromatography and eluted with
ethyl acetate (Rf = 0.3). The required product was obtained as a yellow
solid (0.310 g, 29%), mp > 230 °C. 1H NMR (DMSO-d6): 12.26 (1H,
s), 7.48−7.25 (12H, m), 6.67(2H, br). IR: 1591, 1541, 1479, 1433,
1394, 1323, 1276, 1058, 1028, 869, 767, 690 cm−1 1H NMR (DMSO-
d6): 11.35 (1H, s), 7.36−7.17 (10H, m), 5.74 (2H, s), 2.50 (6H, s).
HRESIMS: calculated for C20H20N5, 330.1713; found, 330.1710.
36a−c,e were similarly prepared.
AUTHOR INFORMATION
■
Corresponding Authors
* (C.J.S.) E-mail: c.j.suckling@strath.ac.uk; Tel.: +44 141 548
2271.
*(M.P.B.) E-mail: michael.barrett@glasgow.ac.uk; Tel.: +44
141 330 6904.
*(W.N.H.) E-mail: w.n.hunter@dundee.ac.uk; Tel.: +44 1382
385745.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
2-Amino-6-{4-[3-(4-morpholinyl)propyl]phenyl}-5-phenyl-
3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one trifluoroacetate
(38). 1-{4-[3-(4-Morpholinyl)propyl]phenyl}-2-phenylethanone. 1-
[4-(3-Chloropropyl)phenyl]-2-phenylethanone (5.50 g, 0.02 mol)
was dissolved in toluene (15 mL, dry), to which was added
morpholine (5.20 g, 0.06 mol) with stirring at room temperature.
The reaction mixture was heated under reflux overnight, and then the
solvent and excess morpholine were removed under reduced pressure.
The crude material obtained was dissolved in ether and extracted with
30% NaOH (aqueous). The organic layer was extracted with water,
dried (Na2SO4), and filtered, and the solvent was removed under
reduced pressure to give the required product as a thick brown oil. The
crude product was applied to a silica gel column chromatography and
eluted with ethyl acetate to give pale yellow thick oil (6.325 g, 98%).
1H NMR as HCl salt (DMSO-d6): 8.00 (2H, d, J = 8.2 Hz), 7.41 (2H,
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support was provided by the Medical Research
Council (UK) through grant no. G0901426/1 and the
Wellcome Trust, grant nos. 094090 and 100476. We thank
Craig Irving, Patricia Keating, Gavin Blackburn, and Gavin Bain
for their help and support, which they provided during the
course of this research work, and Blair Johnston for advice in
the preparation of the manuscript.
ABBREVIATIONS USED
d, J = 8.2 Hz), 7.31−7.20 (5H, m), 4.35 (2H, s), 3.92−3.82 (4H, m),
3.39 (2H, d, J = 12.2 Hz), 3.07−3.01 (4H, m), 2.73 (2H, t, J = 7.7 Hz),
2.08 (2H, qt, J = 4.2 Hz). IR: 717, 740, 788, 806, 842, 991, 1180, 1215,
1317, 1356, 1405, 1433, 1567, 1601, 1682 cm−1
■
AQP2, aquaglyceroporin 2; CMM, Creek’s minimal medium;
DHFR, dihydrofolate reductase; HAT, Human African
Trypanosomiasis; HPLC, high-performance liquid chromatog-
raphy; HRCIMS, high-resolution chemical ionization mass
spectroscopy; HREIMS, high-resolution electron ionization
mass spectroscopy; HRESIMS, high-resolution electrospray
ionization mass spectroscopy; HRFABMS, high-resolution fast
atom bombardment mass spectroscopy; KO, knockout; MTX,
methotrexate; PTR1, pteridine reductase 1; TbPTR1, Trypa-
nosoma brucei pteridine reductase 1
2-Bromo-1-{4-[3-(4-morpholinyl)propyl]phenyl}-2-phenyletha-
none. 1-{4-[3-(4-Morpholinyl)propyl]phenyl}-2-phenylethanone
(0.900 g, 2.783 mmol) was dissolved in chloroform (25 mL).
Hydrobromic acid in acetic acid (33%, 1 mL) was added at room
temperature with stirring. Bromine (0.5 mL) in chloroform (10 mL)
was added to the reaction mixture at room temperature with stirring.
The dropwise addition continued until slight bromine coloration
remained. The stirring was continued for further 30 min. Aqueous
sodium sulfite (10%) solution was added, and the reaction mixture was
then extracted. The organic layer was collected and washed with a
saturated solution of sodium hydrogen carbonate followed by brine,
the organic layer was dried (Na2SO4) and filtered, and the solvent was
removed under reduced pressure to give the required product (1.020
g, 91%) as pale yellow oil. This material was used in the next
experiment without further purification.
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process for the preparation of the key intermediate 2-[2-(4-
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NOTE ADDED AFTER ASAP PUBLICATION
■
The version of this paper that was published ASAP on July 29,
2014, contained incorrect versions of both Scheme 4 and
Scheme 5. The corrected version was reposted July 30, 2014.
P
dx.doi.org/10.1021/jm500483b | J. Med. Chem. XXXX, XXX, XXX−XXX