Pyrimidine-Based Inhibitors of Dynamin I GTPase Activity: Competitive Inhibition at the Pleckstrin Homology Domain

Article abstract of DOI:10.1021/acs.jmedchem.6b01422

The large GTPase dynamin mediates membrane fission during clathrin-mediated endocytosis (CME). The aminopyrimidine compounds were reported to disrupt dynamin localization to the plasma membrane via the PH domain and implicate this mechanism in the inhibition of CME. We have used a computational approach of binding site identification, docking, and interaction energy calculations to design and synthesize a new library of aminopyrimidine analogues targeting site-2 of the pleckstrin homology (PH) domain. The optimized analogues showed low micromolar inhibition against both dynamin I (IC₅₀ = 10.6 ± 1.3 to 1.6 ± 0.3 μM) and CME (IC_50(CME) = 65.9 ± 7.7 to 3.7 ± 1.1 mM), which makes this series among the more potent inhibitors of dynamin and CME yet reported. In CME and growth inhibition cell-based assays, the data obtained was consistent with dynamin inhibition. CEREP ExpresS profiling identified off-target effects at the cholecystokinin, dopamine D₂, histamine H₁ and H₂, melanocortin, melatonin, muscarinic M₁ and M₃, neurokinin, opioid KOP and serotonin receptors.

Full text of DOI:10.1021/acs.jmedchem.6b01422

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Article
Pyrimidine-based inhibitors of Dynamin I GTPase activity:
Competitive inhibition at the pleckstrin homology domain
Luke R. Odell, Mohammed K. Abdel-Hamid, Timothy A Hill, Ngoc Chau, Kelly A Young, Fiona M.
Deane, Jennette A. Sakoff, Sofia Andersson, James A Daniel, Phillip J Robinson, and Adam McCluskey
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01422 • Publication Date (Web): 07 Dec 2016
Downloaded from http://pubs.acs.org on December 12, 2016
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Pyrimidine-based inhibitors of Dynamin I GTPase activity:
Competitive inhibition at the pleckstrin homology domain
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†
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Luke R. Odell, Mohammed K. AbdelꢀHamid, Timothy A. Hill, Ngoc Chau, Kelly A.
1
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3,§
Young, Fiona M Deane, Jennette A Sakoff, Sofia Andersson, James A. Daniel, Phillip J.
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1*
Robinson, and Adam McCluskey
1
Chemistry, School of Environmental and Life Sciences, The University of Newcastle,
Callaghan, NSW 2308, Australia.
2
Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526,
Egypt.
3
Children’s Medical Research Institute, 214 Hawkesbury Road, The University of Sydney,
214 Hawkesbury Road, Westmead NSW 2145, Australia.
4
Experimental Therapeutics Group, Department of Medical Oncology, Calvary Mater
Newcastle Hospital, Edith Street, Waratah, 2298, NSW, Australia.
5
Department of Biology and Chemical Engineering, Mälardalens University, Box 325, Sꢀ631
0
5, Eskilstuna, Sweden.
†
Present address: Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry,
Uppsala Biomedical Centre, Uppsala University, Box 574, SEꢀ751 23 Uppsala, Sweden.
§
Present address: Molecular Neurobiology Group, Max Planck Institute of Experimental
Medicine, HermannꢀRein Str. 3 Göttingen 37075, Germany.
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Abstract
The large GTPase dynamin mediates membrane fission during clathrinꢀmediated
endocytosis (CME). The aminopyrimidine compounds were reported to disrupt dynamin
localization to the plasma membrane via the PH domain and implicate this mechanism in the
inhibition of CME. We have used a computational approach of binding site identification,
docking and interaction energy calculations to design and synthesise a new library of
aminopyrimidine analogues targeting Siteꢀ2 of the pleckstrin homology (PH) domain. The
optimised analogues showed low micromolar inhibition against both dynamin I (IC50 = 10.6 ±
1.3 – 1.6 ± 0.3 µM) and CME (IC50(CME) = 65.9 ± 7.7 – 3.7 ± 1.1 ꢀM) which makes this series
among the more potent inhibitors of dynamin and CME yet reported. In CME and growth
inhibition cell based assays the data obtained was consistent with dynamin inhibition.
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CEREP ExpresS profiling identified offꢀtarget effects at the cholecystokinin, dopamine D
histamine H and H , melanocortin, melatonin, muscarinic M and M , neurokinin, opioid
KOP, serotonin and dopamine receptors.
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,
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Introduction
The dynamins I, II and III (dynI, dynII and dynIII, respectively) are the products of three
genes and belong to the superfamily of large (~100 kDa) GTPꢀbinding proteins. These
enzymes mediate membrane fission during clathrinꢀmediated endocytosis (CME), and play
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1
ꢀ3
additional cellular roles in the exocytic fusion pore, actin dynamics and cytokinesis.
A
4
5, 6
role for dynamin in the sperm acrosome reaction, kidney diseases, bone loss
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9,10
diseases and in the spread of infectious diseases has been demonstrated.
DynI in
mammals is highly enriched in neurons and chromaffin cells. DynI plays a critical role in
synaptic vesicle endocytosis (SVE) and has been implicated as a potential drug target for
1
1, 12
1, 3
epilepsy.
DynII is ubiquitously expressed, and dynIII is tissueꢀspecific.
The proteins
each are composed of five modular domains: an amino terminal G domain responsible for
binding and hydrolysing GTP, a middle domain involved in self assembly and
oligomerization, a pleckstrin homology (PH) domain which binds to phosphoinositide lipids
of the plasma membrane, a bundle signalling element which is thought to be involved in selfꢀ
assembly and a prolineꢀ and arginineꢀ rich domain (PRD) which interacts with the SH3
13ꢀ18
domains in accessory proteins.
Dynamin inhibitors provide valuable tools for the molecular investigation of
9
endocytic pathways. Deficiencies in endocytic pathways are associated with many
1
9
20
human pathological conditions such as Alzheimer’s disease, Huntington’s disease,
Stiffꢀperson syndrome, Lewy body dementias and CharcotꢀMarieꢀTooth disease and
19ꢀ22
autosomal dominant centronuclear myopathy.
We have reported a broad array of
10, 23
24, 25
dynamin inhibitors including long chain ammonium salts,
bisꢀtryphostins,
room
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temperature ionic liquids, iminochromene, phthalic imides, hydrazides, rhodanines,
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indoles,
quinones, and naphthylimides. Other groups have reported dynasore
3
5ꢀ37
analogues and some selective serotonin reuptake inhibitors as dynamin inhibitors.
Our
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pyrimidineꢀbased derivatives inhibit the lipidꢀstimulated GTPase activity of full length dynI
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and dynII. The reported analogues were found to reversibly inhibit CME in a number of
nonꢀneuronal cell lines, as well as inhibiting SVE in nerve terminals. Further investigations
showed that Pyrimidynꢀ6 (1) and Pyrimidynꢀ7 (2) (Figure 1) disrupt dynamin localization to
the plasma membrane via the PH domain and implicate this mechanism in the blockade of
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CME. Small molecules not directly based on phosphatidylinositol analogues
or
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peptides like triciribine or terreic acid that bind to PH domains are rare and mainly based on
42ꢀ45
the presence of a phosphate or sulphate group that mimics that of phosphatidylinositol.
Herein, we report our development of a new library of aminopyrimidineꢀbased analogues
guided by a computational approach using docking and binding energy calculations based on
4
6
the molecular mechanics/generalized Born volume integration (MM/GBVI) model.
Figure 1. Chemical structures for the dynamin inhibitors Pyrimidynꢀ6 (1) and Pyrimidynꢀ7 (2).
Results and Discussion
The dynamin PH domain consists of a βꢀsheet core (β1ꢀβ7 strands) connected via six loops
47, 48
and capped by an amphipathic Cꢀterminal αꢀhelix.
The binding potentials for small
molecules at the surface of the PH domain were investigated through calculating possible
49
binding sites using MOEꢀSiteFinder software. The investigations identified three potential
binding sites (Figure 2). Siteꢀ1 is located in the grove between the variable loops V1 and V2,
Siteꢀ2 occupies parts of the αꢀhelix as well as β1 and β7 strands, while Siteꢀ3 is positioned in
a small grove between β5 and β6 strands. It is worth mentioning that studies conducted on the
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PH domain of AKT enzyme proposed a small molecule binding pocket that is analogous to
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Siteꢀ3 or parts of Siteꢀ2
of the dynI PH domain. However, the reported compounds
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were either phosphate
or sulphate
bearing molecules which are significantly different
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from the developed aminopyrimidineꢀbased analogues in the current study. An initial shapeꢀ
based docking analysis for 1 into the three potential sites showed a preferential clustering of
docked poses at binding Siteꢀ2 (~ 96 % versus 3% and 0.7% at sitesꢀ1 and ꢀ3 respectively;
Supporting Information, Figure S1) supporting our targeting of Siteꢀ2 for subsequent
modelling steps.
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Figure 2. A protein backbone representation of the dynI PH domain showing potential
smallꢀmolecule binding sites identified using the MOEꢀSiteFinder software as colour
coded spheres.
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A second docking of 2 into Siteꢀ2 was performed using a flexible docking approach in
which amino acids within 4 Å of the calculated binding spheres were defined as flexible
residues (Experimental). The resulting complexes were subjected to simulated annealing
(heating to 700 K over 5,000 steps, followed by cooling to 300 K over 10,000 steps) with the
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energy minimization steps performed using the CHARMm force field. The highest ranked
binding pose of 2 with the PH domain was extracted and subjected to further binding
potential interaction and energy calculations. The docked 2 positions the alkyl side chain
within the hydrophobic grove formed by Ile524, Val571, Ile581, Phe582 and Leu599 and
Leu601 (Figure 3). In this configuration, 2 projects the aminopyrimidine moiety into a
hydrophilic box lined by Arg518, Glu607, Asp608 and Ser611. Three key hydrogen bond
interactions were identified which involved the backbone carbonyl of Trp521 interacting with
the C4ꢀNH moiety, the side chain of Arg518 donating a hydrogen bond to the terminal N,Nꢀ
dimethylamine group and the side chain of Asp608 donating a hydrogen bond to the C2ꢀNH
moiety.
Figure 3. A representation of the predicted binding pose of 2 (stick, colour coded by
atom type) binding within Siteꢀ2 of the dynI PH domain (selected residues and
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backbone are shown and labelled). Surface representation; red: exposed, green: cavity.
The overlayed electrostatic map shows alignment of the alkyl side chain of 2 into
hydrophobic grove (green mesh) formed by Ile524, Val571, Ile581, Phe582 and
Leu599 and Leu601. The aminopyrimidine moiety projects towards the hydrophilic
region formed by Arg518, Glu607, Asp608 and Ser611. Potential hydrogen bond
interactions are shown as dashed lines.
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The MM/GBVI calculations began with the replacement of the N,Nꢀdimethylethaneꢀ1,2ꢀ
amine moiety with hydrogen, chloro, propyl and ethaneꢀ1,2ꢀdiamine substituents.
Calculations of the binding energy difference (ꢁꢁG ) predicted that hydrogen and propyl
b
substituents would exhibit lower binding at the PH domain with ꢁꢁG
kcal/mol and +4.3 kcal/mol, respectively. Calculation of ꢁꢁG for the chloro moiety also
showed a predicted lower binding at +1.6 kcal/mol. Whereas, the ꢁꢁG for
ethylethylenediamine moiety predicted a modest increase in activity with a predicted value of
1.3 kcal/mol, indicative of favourable interactions. These results further highlight the
b
values of +7.2
b
b
ꢀ
importance of hydrogen bonding for the inhibitory activity, as observed with the docked 2.
The modelling analysis was also valid for the scaffold based on 1 (data not shown).
Accordingly, a small library of pyrimidine based analogues with selected modifications at C2
(
for 2-based analogues) and C4 (for 1ꢀbased analogues) was designed and synthesised.
Libraries were assembled by treatment of 2,4ꢀdichloroꢀ6ꢀmethylpyrimidine (3) with
dodecylamine at room temperature in THF followed by chromatographic separation
Supporting Information) to afford the monosubstituted aminopyrimidines 4a and 4b in 44%
(
and 35% respectively (Scheme 1). Treatment of 4a and 4b, separately, with five equivalents
of selected amines (see Table 1 for detail) in butanol at 130 ºC under sealed tube conditions
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afforded the desired diaminated pyrimidines (5a – 9b) in poor (17%) to excellent (96%)
yields.
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Scheme 1: Reagents and Conditions: (i) dodecylamine, THF, 30°C, 18 h, chromatographic
separation, (ii) alkylamine, nꢀBuOH sealed tube, 130°C, 18 h
.
The synthesised pyrimidines (4a–9a and 4b-9b) were evaluated for their ability to inhibit
dynI GTP hydrolysis activity stimulated by phosphatidylserine (PS) liposomes (Table 1).
Table 1. Inhibition PSꢀstimulated dyn I GTPase activity by aminopyrimidines 1, 2, 4a–9a
and 4b-9b.
DynI
DynI
IC50 (µM)
1.4 ± 0.2
26.6 ± 9.3
Compound
R
Compound
R
IC50 (µM)
1.1 ± 0.05
32.5 ± 4.5
a
b
2
NHCH
2
CH
2
NH(CH
3
)
2
1
NHCH
Cl
2
2
CH
2
2
3 2
NH(CH )
Cl
4b
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5
6
a
a
a
NHCH
NHCH
NHCH
CH
CH
CH
CH
61.8 ± 9.4
5.3 ± 0.1
4.6 ± 0.5
5b
6b
7b
NHCH
CH
CH
NH
OH
4.7 ± 0.8
18.6 ± 4.3
5.5 ± 0.7
2
2
2
2
2
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3
3
NH
OH
2
NH
2
CH
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CH
2
2
NHCH
2
CH
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NHCH
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(CH
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N(CH
3
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2
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14 ± 1.6
8b
9b
NHCH
N(CH
2
(CH
2
)
2
N(CH
3
)
2
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3.3 ± 0.05
8
a
a
N(CH
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)
2
3 2
)
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a
b
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Pyrimidynꢀ7, Pyrimidynꢀ6 (from ).
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Replacement of N,Nꢀdimethylethaneꢀ1,2ꢀamine moiety by a chloro moiety produced
analogues with moderate activity (4a,b). The omission of the terminal dimethylamino moiety
led to decreased potency for the C2 analogue 5a, yet was unexpectedly tolerated for the C4
analogue 5b. The extension of the N,Nꢀdimethylethaneꢀ1,2ꢀamine moiety by a single
methylene resulted in an activity reduction (8a,b), while contraction to N,Nꢀdimethylamine
(
9a,b) was generally tolerated with 9b showing unexpected high potency (IC = 3.3 ± 0.05
50
µM). Consistent with the modelling results, short terminally functionalized amines were well
tolerated, viz the ethylenediamine substituted 6a,b and the aminoethanolamine substituted
7
a,b.
Although the monochloro analogues 4a and 4b showed only modest activity, their ease of
synthetic access was encouraging for the synthesis of a library of monoꢀaminated
chloropyrimidines with different aminoalkyl tails. The synthesis was conducted as per
Scheme 1 on treatment of 3 with an array of simple alkyl amines to afford two analogue
libraries (after chromatographic separation), 10aꢀ26a and 10bꢀ26b in poor (19%) to good
(
53%) yields for each C2ꢀ and C4ꢀ aminopyrimidine isomer. In keeping with the predicted
critical nature of the Hꢀbonding in this compound class, none of these simple alkyl
aminopyrimidine analogues were active against dynI at concentrations up to 300 µM
(
Supporting Information, Table S1).
Since the N,Nꢀdimethylethaneꢀ1,2ꢀamine substituted pyrimidines showed the best activity
among the analogues in Table 1, we next explored possible modifications to the alkyl tail.
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MM/GBVI calculations were executed for replacing the dodecyl tail of 2 with H, methyl and
octyl groups. The resultant ꢁꢁG
b
values were +9.43, +4.64, and −1.65 kcal/mol,
respectively. This was consistent with the docking results and confirmed the crucial role of
hydrophobic interactions for the binding of this type of compounds with the PH domain. In
addition, the role of the methyl substituent C6 was tested computationally and showed a nonꢀ
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significant contribution in binding with ꢁꢁG
value of only −0.35 kcal/mol.
b
Accordingly, a library of ꢀdimethylethylenediamine substituted pyrimidines was
N,N
synthesized using the same general procedure in Scheme 1. The parent monoꢀaminated
chloropyrimidine (10a – 26a and 10b-26b, Supplementary Information) was treated with five
equivalents of N,N-dimethylethylenediamine to afford the desired disubstituted pyrimidines
(27a – 39a and 27b – 39b) in poor (19%) to excellent (93%) yields. A subꢀlibrary (40a,b;
41a,b; 42 and 43) of selected desꢀmethyl analogues was also prepared. The synthesised
derivatives were screened for their ability to inhibit dynI GTPase activity (Table 2).
Table 2. Inhibition PSꢀstimulated dyn I GTPase activity by aminopyrimidines (27aꢀ41a,
27b-41b, 42 and 43).
R
N
N
HN
N
Dyn I
Dyn I
Compound
R
Compound
R
IC50 (µM)
IC50 (µM)
a
a
2
2
2
3
7a
8a
9a
0a
NHCH
2
CH
3
ꢀ
27b
28b
29b
30b
31b
NHCH
2
CH
3
ꢀ
a
a
NHCH
NHCH (CH
CH
CH
CH
ꢀ
NHCH
CH
CH
ꢀ
2
2
3
2
2
3
b
a
2
2
)
2
3
> 50
NHCH
NHCH
NHCH
2
2
2
(CH
(CH
(CH
2
)
)
)
2
4
6
CH
CH
CH
3
3
3
ꢀ
NHCH
(CH
(CH
)
)
CH
CH
10.6 ± 1.3
1.6 ± 0.3
5.8 ± 0.7
2.5 ± 1.3
2
2
2
2
4
6
3
3
2
2
31a
NHCH
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32a
NHCH
2
(CH
2
)
8
CH
3
3.8 ± 0.7
3.2 ± 0.6
8.4 ± 0.8
32b
33b
34b
35b
36b
NHCH
2
(CH
2
)
8
CH
3
9.3 ± 0.9
Not active
4.4 ± 0.5
33a
34a
35a
36a
NHCH
NHCH
2
(CH
(CH
2
)
)
12CH
16CH
3
3
NHCH
NHCH
2
(CH
(CH
2
)
)
12CH
16CH
3
2
2
2
2
3
a
b
NHCH(CH
N(CH (CH
3
)CH
2
CH
3
ꢀ
NHCH(CH
N(CH (CH
3
)CH
2
CH
3
> 50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
)
8
CH
3
)
2
7.5 ± 1.5
2
2
)
8
CH
3
)
2
5.2 ± 0.5
3
7a
8a
20.6 ± 3.0
37b
3.8 ± 0.8
b
b
3
> 50
38b
39b
> 50
39a
52.0 ± 4.2
59.8 ± 5.5
4
0a
1a
NHCH
2
(CH
2
)
8
CH
3
2.9 ± 0.3
1.8 ± 0.3
40b
41b
NHCH
2
(CH
2
)
8
CH
3
44.9 ± 3.5
2.0 ± 0.3
4
NHCH
2
(CH
2
)
10CH
3
NHCH
2
(CH
2
)
10CH
3
4
2
NHCH
2
(CH
2
)
8
CH
3
5.1 ± 0.5
43
NHCH
2
(CH
2
)
10CH
3
7.7 ± 1.4
a
b
no activity at 300 µM drug concentration; ~ 50% activity at 300 µM drug concentration.
The data in Table 2 shows that introduction of short alkyl amines such as ethyl to
n
ꢀbutyl
or benzyl moieties (27a-29a, 27b-29b and 38a,b) is detrimental to dynI activity, with these
analogues being inactive. Increasing the alkyl chain length to ꢀhexyl dramatically increased
n
potency (IC50 of 10.6 ± 1.3 and 5.8 ± 0.7 µM for 30a and 30b respectively), while chain
lengths of octyl to octadecyl were essentially equipotent (31a-34a and 31b-34b), with the
exception that 33b was inactive. Branching or cyclization of the alkyl chain had little to no
effect on the inhibition activity (36a,b; 37a,b versus 31a-33a and 31b-33b), indicating the
binding pocket at this side chain has a high hydrophobic and steric tolerance. This is
consistent with our docking poses and calculated binding energies. However, the
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
introduction of a
N
ꢀ(cyclopropylmethyl)propanꢀ1ꢀamine moiety (39a,b) produced only
modest levels of activity. The corresponding C6ꢀdesmethyl analogues (40a – 41b, 42 and 43)
with the exception of 40b, displayed excellent dynamin inhibition (relative to the current
2
3ꢀ38
published dynamin inhibitors)
and confirmed no role for the methyl group for binding
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
with the PH pocket. This was confirmed by evaluation of the energy minimised docked pose
of 42 as a representative desꢀmethyl analogue. Within Site 2, analogue 42 showed a similar
orientation to 2 at the binding site (Figure 4), except that the 42ꢀTrp521 hydrogen bond is
formed between one of the pyrimidine nitrogen atoms to the backbone of Trp521 which with
1
is formed from the C4ꢀNH moiety to the Trp521 carbonyl moiety (Supplementary
Information, Figure S2). The only exception being the hydrogen bonding with Trp512 which
in the case of 42.
Asp608
Ser525
Ser611
Arg518
Cys603
Thr523
Trp521
Figure 4. Overlap of the predicted binding poses for analogues 2 (stick, nonꢀcarbon atoms
green) and 42 (stick, nonꢀcarbon atoms pink) within the Siteꢀ2 binding site of dynI PH
domain (selected residues and backbone are shown and labelled). The two analogues are
showing similar orientation with the potential of forming same hydrophobic and hydrogen
bond interactions at the binding site.
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Given the role of dynamin in CME we examined the activity of selected analogues against
32
dynII and/or CME activity in U2OS osteosarcoma cells, with this data presented in Table 3.
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Inhibition of PSꢀstimulated dynII GTPase and clathrinꢀmediated endocytosis
(
CME) by selected aminopyrimidine analogues.
DynI
Compound
DynII
CME
IC50 (µM)
IC50 (µM)
1.3 ± 0.4
4.0 ± 2.2
IC50 (µM)
37.8 ± 9.7
31.0 ± 3.8
47.4 ± 12.4
8.7 ± 1.8
10.3 ± 2.5
5.1 ± 1.2
12.9 ± 1.4
65.9 ± 7.7
6.1 ± 1.1
3.7 ± 1.1
6.0 ± 0.7
8.5 ± 1.4
9
b
3.3 ± 0.05
10.6 ± 1.3
5.8 ± 0.7
1.6 ± 0.3
2.5 ± 1.3
3.8 ± 0.7
3.2 ± 0.6
3.8 ± 0.8
1.8 ± 0.3
2.9 ± 0.3
5.1 ± 0.5
7.7 ± 1.4
3
0a
0b
31a
1b
a
3
ND
ND
ND
3
3
3
2a
3a
1.6 ± 0.4
ND
37b
1.8 ± 0.8
ND
4
4
1a
0a
ND
4
2
1.8 ± 0.4
8.3 ± 2.2
43
a
ND = Not determined
Comparing the inhibition data for dynI and dynII activities indicates no significant
selectivity for these aminopyrimidine based dynamin inhibitors against the two dynamin
isoforms. Most of the tested analogues potently inhibited CME, which is mediated by dynII,
at a similar order of potency in which they inhibited dynamin. Aminopyrimidines 32a, 40a
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and 42 (IC50(CME) = 5.1 ± 1.2, 3.7 ± 1.1, 6.0 ± 0.7
CME inhibitors reported to date.
ꢀM respectively) are among the more potent
The aminoalkyl moiety of the pyrimidine analogues reported herein has the potential to
bind to other molecular targets introducing undesirable offꢀtarget effects. To this end we
examined the dynamin active 1 and a dynamin inactive analogue, 15a (Supplementary
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Information, dyn I IC50 >300
ꢀM) in a series of twelve cancer derived cell lines was
5
0
undertaken and these data are presented in Table 4.
Table 4. Growth inhibition (GI50
ꢀM) values of pyrimidine analogues 1 and 15a against a
panel of human cancer cell lines. Values in italics (for compound 15a) represent the
percentage cell death at 25
ꢀM compound concentration.
Cell Line
a
a
b
c
d
b
SW480
HT29
SMA
MCF-7
BE2-C
SJ-G2
Compound
0
.8 ± 0.03
1.4 ± 0.1 1.3 ± 0.2 1.5 ± 0.0 1.5 ± 0.0
1.5 ± 0.0
1
3
7 ± 2
56 ± 1
65 ± 2
53 ± 2
46 ± 2
25 ± 3
15a
Cell Line
Compound
1
e
f
g
h
b
i
MiaPaCa-2
A2780
A431
H460
U87
Du145
1.5 ± 0.0
1.8 ± 0.5 1.7 ± 0.2 2.5 ± 0.2 1.5 ± 0.0
1.9 ± 0.1
15a
42 ± 4
63 ± 1
25 ± 1
21 ± 3
21 ± 2
42 ± 3
a
b
c
d
e
f
g
h
i
Colon, Glioblastoma, Breast, Neuroblastoma, Pancreas, Ovarian, Skin, Lung, Prostate
cancer derived cell lines.
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Evaluation of the growth inhibition data (Table 4) reveals high levels of growth inhibition
across all 12 cell lines examined with the dynamin active 1, but low levels of growth
inhibition with dynamin inactive 15a. This is consistent with the known effects of dynamin
51,52
inhibition in cell growth assay.
Importantly, the retention of the dodecylamino moiety
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16
17
18
19
20
21
22
23
24
25
26
27
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29
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
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with 15a supports, at least in this phenotypic assay, limited off target effects within this
compound class.
Further exploration of the potential off target effects of 1 via CEREP ExpresS profiling at
10 ꢀM was used to assess drug development suitability. Most of the 55 binding assays in the
ExpresS Profile panel are human recombinant receptors but includes seven key ion channels
important in synaptic transmission and three transporters. This screening panel identified
2 1 2
activity at the cholecystokinin, dopamine D , histamine H and H , melanocortin, melatonin,
muscarinic M and M , neurokinin, opioid KOP, serotonin and dopamine receptors (Table 5
1
3
and Supplementary Information, Table S2).
Table 5. CEREP ExpresS Panel Screening of 1 at 10
ꢀM drug concentration.
%
Inhibition of
IC50
K
i
Control Specific
Binding
Reference
Compound
Ref
Ref
Assay
(nM)
(nM)
a
Dopamine D2S (h)
99
(+)ꢀbutaclamol
0.81
0.27
a
H1 (h)
102
106
pyrilamine
cimetidine
1.9
0.71
490
a
H2 (h)
500
NDPꢀalpha ꢀ
MSH
c
MC4 (h)
96
91
0.16
27
0.14
24
a
M1 (h)
pirenzepine
4ꢀDAMP
a
M3 (h)
104
0.31
0.22
[
Nleu10]ꢀNKA
(4ꢀ10)
c
NK2 (h)
93
97
3.6
2.0
c
kappa (KOP)
U 50488
0.68
0.45
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1
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1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
c
c
5ꢀHT2A (h)
5ꢀHT2B (h)
93
94
99
91
98
ketanserin
0.52
3.2
0.28
1.6
(±)DOI
serotonin
serotonin
serotonin
c
5
5
5
ꢀHT5a (h)
260
130
0.47
130
61
c
ꢀHT6 (h)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
c
ꢀHT7 (h)
0.17
norepinephrine transporter
a
(h)
96
protriptyline
BTCP
2.9
12
2.1
6.2
a
dopamine transporter (h)
105
a
b
antagonist radioꢀligand; nonꢀselective, antagonist radioꢀligand
5
3ꢀ55
While most drugs display a degree of polypharmacology,
the activity of
aminopyrimidine 1 against the targets identified in Table 5 suggests a potentially limiting
degree of offꢀtarget interactions. Ultimately, in vivo evaluation will be the determinant of
safety and tolerability. The current single dose CEREP ExpresS panel analysis suggests a
current upper limit of a 10ꢀfold dynamin selectivity. Clearly these identified off target effects
require careful evaluation and monitoring during the onꢀgoing development of this family of
dynamin inhibitors.
Conclusions
MOEꢀSiteFinder identified three potential binding sites for 1 within dynamin’s PH
domain. Shape based clustering showed a marked preference for docking within Siteꢀ2 at
9
6% vs 3% and 0.7% at Sites 2, 1 and 3 respectively. Molecular dynamics revealed three key
hydrogen bond interactions: the carbonyl of Trp521 interacting with the C4ꢀNH moiety, the
side chain of Arg518 donating a hydrogen bond to the terminal N,Nꢀdimethylamine group
and the side chain of Asp608 donating a hydrogen bond to the C2ꢀNH moiety. Modelling of
the desꢀmethyl analogues in Siteꢀ2 revealed no major differences in binding pose or
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interactions supporting limited involvement of the methyl moiety in binding. MM/GBVI
calculations were used to guide subsequent focused compound library development.
Subsequent synthesis identified a wide range of aminopyrimidineꢀbased analogues returning
dynI IC50 values <10 ꢀM. The introduction of long alkyl chains was favoured, which was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
consistent with our docking poses and calculated binding energies. Analogues 32a, 37b, 42
and 43 were equipotent against dynI and II, and all analogues examined for their ability to
block dynaminꢀdependent CME were also potent GTPase inhibitors with IC50 values,
consistent with their activity against isolated dynamin. These pyrimidine analogues are
amongst the more potent inhibitors of CME reported. The data is consistent with the
hypothesis that these compounds interact with the PH domain of dynamin. This study
suggests a pathway for the future design other PH domain targeted nonꢀphosphate/nonꢀ
sulphate based small molecules that might exhibit specificity for different PH domains.
In CME and growth inhibition cell based assays the data obtained was consistent with
dynamin inhibition. CEREP ExpresS profiling identified activity at the cholecystokinin,
2 1 2 1 3
dopamine D , histamine H and H , melanocortin, melatonin, muscarinic M and M ,
neurokinin, opioid KOP, serotonin and dopamine receptors. When considered in combination
with our identified modelling led approach and consistent dynamin inhibition phenotype in
cells it is evident that onꢀgoing development of the aminopyrimidine series of dynamin
inhibitors will require onꢀgoing monitoring of the identified off target effects.
Experimental Section
Molecular Modelling
18
The PH domain was extracted from the crystal structure of dynI (PDB: 3ZVR). Water
and axillary molecules were omitted and the structure was typed with CHARMm force field.
An in vacuo energy minimization procedure was performed, after fixing the protein
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
backbone, using steepest descent algorithm for 2000 steps until an energy convergence of
≤
0.1 kcal/mol Å was obtained. The calculation of potential smallꢀmolecule binding pockets
was performed using Molecular Operating Environment MOEꢀSiteFinder function of the
49
MOE software. Initial docking of 2 was performed using MOEꢀDock using “Triangle
Matcher” as the ligand placement method and binding Sites 1ꢀ3 as the potential binding
pockets (Figure 2). The obtained binding poses were clustered, showing the most abundant
clusters for 2 around the potential binding Siteꢀ2. The second docking step applied the
flexible docking protocol developed by Koska et al. using Accelrys Discovery Studio
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
56ꢀ58
software.
An initial step of generating side chain conformations for amino acid residues
59
within the binding site was performed using ChiFlex algorithm. Amino acids within 4 Å of
the predicted binding site spheres were defined as flexible residues. This was followed by
docking of 2 into each of the generated protein side chain conformation using LibDock
algorithm. Subsequently, the binding site side chains were refined in the presence of the
59
ligand using ChiRotor algorithm. The final step included simulated annealing (heating to
00 K over 5,000 steps flowed by cooling to 300 K over 10,000 steps) and energy
minimization of each ligand pose under CHARMm force field using CDocker protocol.
7
57
The highest scored complex as defined by the calculated CDocker energy (kcal/mol) was
considered for analysis and interaction energy estimation. The obtained model was analysed
for potential interaction using MOEꢀLigX module. The interaction energy estimation was
calculated based on the molecular mechanics/generalized Born volume integration
46, 60
(MM/GBVI) model using MOE software.
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Biology
Dynamin GTPase assay
Phosphatidylserine (PS), phenylmethylsulfonylfluoride (PMSF) and Tween 80 were from
Sigma Aldrich (St. Louis, MO). GTP was from Roche Applied Science (Basel, Switzerland)
and leupeptin was from Bachem (Bubendorf, Switzerland). All other reagents were of
analytical reagent grade or better.
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11
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13
14
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16
17
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19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Potential inhibitor compounds were made up as stock solutions of 30 mM in 100% DMSO
and further diluted in assay buffer such that the final DMSO concentration was no greater
than 3.3% (dynI GTPase activity was unaffected by DMSO up to this concentration).
Compounds were made up fresh from powder stock just prior to commencing of assay and
stock solutions were stored at ꢀ20˚C for several months.
Native endogenous dynI was purified from sheep brain by extraction from the peripheral
membrane fraction of whole brain, and affinity purification on GSTꢀAmph2ꢀSH3ꢀsepharose
3
8
as previously described, yielding 8ꢀ10 mg of protein from 250 g of sheep brain.
The Malachite Green method was used for the sensitive colorimetric detection of
38
orthophosphate (P
i
) release from GTP as previously described. Briefly, GTPase activity
was stimulated on 20ꢀ30 nM dynI by 4 µg/mL sonicated PS liposomes, in the presence of 1
ꢂg/mL leupeptin, 0.1 mM PMSF, 0.3 mM GTP and test compound for 30 min at 37 °C in a
final assay volume of 150 ꢂL. The reaction was terminated with 10 ꢂL of 0.5 M EDTA (pH
7.4) and phosphate was detected by addition of Malachite Green solution (40 ꢂL: 2% w/v
ammonium molybdate tetrahydrate, 0.15% w/v Malachite Green and 4 M HCl) for 5 min.
Data analysis and IC50 values estimation were calculated in Microsoft Excel and GraphPad
Prism v5.0, using nonꢀlinear regression methods.
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6
CME Assay
Fibronectin and DAPI were from Sigma Aldrich (St. Louis, MO). Paraformaldehyde
PFA) was from BDH (AnalaR Merck Chemicals, Darmstadt, Germany). Phosphate buffered
(
saline (PBS), foetal bovine serum (FBS) and Dulbecco’s Minimal Essential Medium
(DMEM) were from Invitrogen (Paisley, Strathclyde, UK). Alexaꢀ594 conjugated
Transferrin (TfnꢀA594) was from Molecular Probes (Eugene, OR, USA). All other reagents
were of analytical grade or better.
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CME was examined by a high throughput, quantitative assay for measuring transferrin
3
2
(Tfn) uptake in U2OS osteosarcoma cells, based on methods described previously. Briefly,
cells were cultured on fibronectinꢀcoated 96ꢀwell glassꢀbottomed plate, maintained in DMEM
o
supplemented with 10% FBS at 37 C in 5% CO
2
. Cells were then serumꢀstarved overnight
(16 h) before being treated with test compounds or vehicle (1% DMSO) for 30 min at 37˚C.
TfnꢀA594 (4 µg/mL) was applied for 8 min at 37˚C and endocytosis was arrested by placing
o
the cells on ice (4 C). Surfaceꢀbound Tfn was removed with an iceꢀcold acid wash solution
(0.2 M acetic acid, 0.5 M NaCl, pH 2.8) for 10 min followed by an iceꢀcold PBS wash for 5
min. Cells were immediately fixed with 4% PFA for 10 min at room temperature. Nuclei
were labelled with DAPI stain solution. Cell imaging and data analysis were conducted by a
high content imaging system MetaXpress (Molecular Devices). Quantitative analysis of Tfnꢀ
A594 uptake in U2OS cells was performed by high content imaging. Nine images were
collected from each well, averaging 40ꢀ50 cells per image, totalling of ~1,200 cells per
condition. IC50 values were calculated using Graphpad Prism version 5, where data was
expressed as percentage of control cells (vehicle treated).
Growth Inhibition
All test agents were prepared as stock solutions (20 mM) in dimethyl sulfoxide (DMSO)
and stored at ꢀ20 °C. Cell lines used in the study included MCFꢀ7 (breast carcinoma); HT29,
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SW480 (colorectal carcinoma); U87, SJꢀG2, SMA (glioblastoma); A2780 (ovarian
carcinoma); H460 (lung carcinoma); A431 (skin carcinoma); Du145 (prostate carcinoma);
BE2ꢀC (neuroblastoma); and MiaPaCaꢀ2 (pancreatic carcinoma). All cell lines were
incubated in a humidified atmosphere 5 % CO at 37 °C. and maintained in Dulbecco’s
2
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modified Eagle’s medium (DMEM; Sigma, Australia) supplemented with foetal bovine
serum (10 %), sodium pyruvate (10 mM), penicillin (100 IU/mL), streptomycin (100 µg/mL),
and Lꢀglutamine (4 mM). Growth inhibition was determined by plating cells in duplicate in
medium (100 µL) at a density of 2500–4000 cells per well in 96ꢀwell plates. On day 0 (24 h
after plating), when the cells are in logarithmic growth, medium (100 µL) with or without the
test agent was added to each well. After 72 h drug exposure, growth inhibitory effects were
evaluated using the MTT (3ꢀ[4,5ꢀdimethyltiazolꢀ2ꢀyl]ꢀ2,5ꢀdiphenyltetrazolium bromide)
assay and absorbance read at 540 nm. An eightꢀpoint doseꢀresponse curve was produced from
which the GI50 value was calculated, representing the drug concentration at which cell growth
was inhibited by 50 % based on the difference between the optical density values on day 0
5
0
and those at the end of drug exposure.
Chemistry
General methods
THF was freshly distilled from sodium–benzophenone. Flash chromatography was carried
1
13
out using silica gel 200–400 mesh (60 Å). H and C NMR were recorded at 300 MHz and
5 MHz respectively using a Bruker Avance 300 MHz spectrometer in CDCl3, CD OD and
7
3
DMSOꢀd6. GCMS was performed using a Shimadzu GCMSꢀQP2100. The instrument uses a
quadrupole mass spectrometer and detects samples via electron impact ionization (EI). The
University of Wollongong, Australia, Biomolecular Mass Spectrometry Laboratory analyzed
samples for HRMS. The spectra were run on the VG Autospecꢀoaꢀtof tandem high resolution
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mass spectrometer using CI (chemical ionization), with methane as the carrier gas and PFK
(perfluorokerosene) as the reference. Microanalysis was conducted at the Microanalyis Unit
at the Australian National University, Canberra Australia. All samples returned satisfactory
analyses. Compound purity was confirmed by a combination of LCꢀMS (HPLC), micro
and/or high resolution mass spectrometry and NMR analysis. All analogues are ≥ 95%
purity.
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(2-Chloro-6-methylpyrimidin-4-yl)dodecylamine (4a) and (4-chloro-6-methylpyrimidin-
3
8
2
-yl)dodecylamine (4b)
To a solution of 2,4ꢀdichloroꢀ6ꢀmethylpyrimidine 3 (0.65 g, 4.0 mmol) in THF (5 mL) was
added dodecylamine (1.48 g, 8 mmol, 2 eq). The resulting solution was stirred at 30 C for 18
°
h, filtered and concentrated in vacuo. The residue was purified by flash chromatography
using 1:9 ethyl acetate : hexanes to yield the corresponding isomers.
1
(
4a) Yield 545 mg, 44%, mp 46ꢀ48°C; H NMR (CDCl ):
δ
0.77 (t, 3H,
= 6.7 Hz), 5.78 (br s, 1H), 6.25 (s, 1H);
13.7, 21.8, 26.2, 28.7, 28.8 (2 × C), 28.9 (3 × C), 29.0, 31.7, 41.3, 98.7
br), 159.2, 163.7 (br), 165.2.
4b) Yield 436 mg, 35%; mp 55ꢀ56°C; H NMR (CDCl
m, 18H), 1.55 (m, 2H), 2.18 (s, 3H), 3.34 (q, 2H, = 7 Hz), 5.52 (br s, 1H), 6.37 (s, 1H); C
13.3, 22.8, 26.3, 28.6 (2 × C), 28.7 (2 × C), 28.8 (2 × C), 28.9, 29.0, 31.2,
0.6, 108.1, 161.3, 163.2, 164.6.
J = 7.0 Hz), 1.26
3
(
s, 18H), 1.44 (m, 2H), 2.17 (s, 3H), 3.15 (q, 2H, J
13
3
C NMR (CDCl ): δ
(
(
1
(
3
): δ 0.83 (t, 3H, J = 7.2 Hz), 1.48
13
J
NMR (CDCl3):
δ
4
4
2
38
N
-Dodecyl-6-methyl-
N -propylpyrimidine-2,4-diamine (5a)
To a solution of (2ꢀchloroꢀ6ꢀmethylpyrimidinꢀ4ꢀyl)dodecylamine (4a) (0.30 g, 1.0 mmol)
in
n
ꢀBuOH (25 mL) was added propylamine (0.30 g, 5 mmol, 5 eq). The reaction mixture
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was heated at 130
°
C for 18 hr, cooled to room temperature and diluted with EtOAc (25 mL)
and brine (25 mL). The organic layer was washed with brine (2 × 25 mL), 10% NaHCO
3
(2
×
25 mL) dried over MgSO and concentrated in vacuo. The residue was purified by dry
4
6
1
column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
3
target compound.
(5a) Yield 146 mg, 68% pale yellow oil.
1
H NMR (CD
3
OD): 0.71 (t,
J
= 6.9 Hz, 3H), 1.15 (m, 18H), 1.38 (m, 2H), 1.97 (s, 3H),
2.72 (br m, 2H), 3.15 (t,
J
= 6.8 Hz, 2H), 3.26 (t, = 5.7 Hz, 2H), 5.46 (s, 1H).
J
13
C NMR (CD OD): 17.1, 24.2 (br), 26.9, 30.3, 32.5, 32.7, 32.8, 32.9 (3 x C), 33.0, 35.2,
3
44.2, 44.5, 46.5 (br), 96.3 (br), 164.2 (br), 166.0 (br), 167.3 (br).
2
4
38
N
-Dodecyl-6-methyl-
To a solution of (4ꢀchloroꢀ6ꢀmethylpyrimidinꢀ2ꢀyl)dodecylamine (4b) (0.3 g, 1.0 mmol) in
ꢀBuOH (25 mL) was added propylamine (0.3 g, 5 mmol, 5 eq). The reaction mixture was
heated at 130 C for 18 hr, cooled to room temperature and diluted with EtOAc (25 mL) and
brine (25 mL). The organic layer was washed with brine (2 × 25 mL), 10% NaHCO (2 × 25
mL) dried over MgSO and concentrated in vacuo. The residue was purified by dry column
N -propylpyrimidine-2,4-diamine (5b)
n
°
3
4
flash chromatography using EtOAc followed by 1:1 CHCl
3 3
:CH OH to yield the target
compound.
(5b) Yield 146 mg, 68% pale yellow oil.
1
3
H NMR (CDCl ): δ 0.83 (t, 3H, J = 6.9 Hz), 0.89 (t, 3H, J = 7.4 Hz), 1.26 (s, 18H), 1.50
(m, 4H), 2.09 (s, 3H), 3.23 (m, 4H), 4.54 (br m, 2H), 5.51 (s, 1H);
1
3
C NMR (CDCl
3
): δ 10.8, 13.3, 22.1, 24.1, 23.4, 26.4, 28.7, 28.9 (3 × C), 29.0 (2 × C),
29.5, 31.3, 40.7, 42.5, 91.7 (br), 161.2, 163.2, 163.8 (br).
2
4
N
-(2-Aminoethyl)-
N
-dodecyl-6-methylpyrimidine-2,4-diamine (6a)
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6
Synthesized using the general procedure as for 5a from 4ꢀchloroꢀNꢀdodecylꢀ6ꢀ
methylpyrimidinꢀ2ꢀamine (4a) and 1,2ꢀdiaminoethane. The residue was purified by dry
3 3
column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield the
target compound.
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1
(6a) Yield 174 mg, 82% pale yellow oil; H NMR (CD OD): δ 0.88 (t, 3H,
J
= 7.3 Hz),
= 7.0 Hz),
OD):δ 12.5, 21.3, 21.7, 25.9 (br), 26.2,
3
1
3
2
.27 (s, 18H), 1.54 (m, 4H), 2.09 (s, 3H), 2.87 (t, 2H, J = 5.8 Hz), 3.27 (t, 2H, J
13
.43 (t, 2H,
J
= 6.0 Hz), 5.64 (s, 1H); C NMR (CD
3
8.5, 28.6, 28.7, 28.8 (2 x C), 30.6 (br) 31.3, 39.9 (br), 40.5, 42.4 (br), 92.6 (br), 161.6, 163.0
(
br), 163.4; HRMS calculated for C H N , 335.3049; found 335.3055.
19 37 5
4
2
N
-(2-Aminoethyl)-
Synthesized using the general procedure as for 5a from 2ꢀchloroꢀ
methylpyrimidinꢀ4ꢀamine (4b) and 1,2ꢀdiaminoethane. The residue was purified by dry
N
-dodecyl-6-methylpyrimidine-2,4-diamine (6b)
N
ꢀdodecylꢀ6ꢀ
3 3
column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield the
target compound.
1
(
6b) Yield 204 mg, 96%, mp 62ꢀ64°C; H NMR (CD OD): δ 0.71 (t, 3H,
J
= 6.9 Hz), 1.15
= 6.8 Hz), 3.26 (t, 2H,
OD): δ 17.1, 24.2 (br), 26.9, 30.3, 32.5, 32.7, 32.8,
2.9 (3 x C), 33.0, 35.2, 44.2, 44.5, 46.5 (br), 96.3 (br), 164.2 (br), 166.0 (br), 167.3 (br);
HRMS calculated for C H N , 335.3049; found 335.3047
3
(
m, 18H), 1.38 (m, 2H), 1.97 (s, 3H), 2.72 (br m, 2H), 3.15 (t, 2H,
J
J
13
= 5.7 Hz), 5.46 (s, 1H); C NMR (CD
3
3
1
9
37
5
2
-(4-Dodecylamino-6-methylpyrimidin-2-ylamino)ethanol (7a)
Synthesized using the general procedure as for 5a from 4ꢀchloroꢀNꢀdodecylꢀ6ꢀ
methylpyrimidinꢀ2ꢀamine (4a) and aminoethanol. The residue was purified by dry column
3 3
flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield the target
compound.
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(7a) Yield 210 mg, 96%, mp 64ꢀ66°C; H NMR (CDCl
3
): δ 0.83 (t, 3H,
J
= 7.0 Hz), 1.22
(
m, 18H), 1.47 (m, 2H), 2.09 (s, 3H), 3.16 (q, 2H,
t, 2H,
J
= 5.9 Hz), 3.41 (t, 2H, J = 4.5 Hz), 3.69
13
(
J
= 4.5 Hz), 5.03 (br s, 1H), 5.50 (s, 1H), 5.66 (br s, 1H); C NMR (CDCl
3
): δ 13.3,
2
2.0, 23.4, 26.3, 26.9, 28.6, 28.7, 28.8, 28.9, 29.0 (2 x C), 31.2, 40.7, 44.1, 63.0, 92.1 (br),
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11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
162.0, 163.1, 164.3 (br); HRMS calculated for C H N O, 336.2889; found 336.2881.
1
9
36
4
2-(2-Dodecylamino-6-methylpyrimidin-4-ylamino)ethanol (7b)
Synthesized using the general procedure as for 5a from 2ꢀchloroꢀNꢀdodecylꢀ6ꢀ
methylpyrimidinꢀ4ꢀamine (4b) and aminoethanol. The residue was purified by dry column
flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield the target
3
3
compound.
7b) yield 167 mg, 77%, 40ꢀ42°C; H NMR (CDCl
18H), 1.47 (m, 2H), 2.09 (s, 3H), 3.27 (t, 2H, = 6.4 Hz), 3.40 (q, 2H,
1
(
3
): δ 0.84 (t, 3H,
J
= 6.9 Hz), 1.28 (m,
= 4.9 Hz), 3.70 (t,
= 4.8 Hz), 4.86 (br s, 1H), 5.26 (br s, 1H), 5.50 (s, 1H); C NMR (CDCl ): δ 13.3,
J
J
13
2
2
1
H,
J
3
2.0, 23.5, 26.4, 28.6, 28.8 (2 x C), 28.9 (2 x C), 29.0, 29.2, 31.3, 40.8, 43.3, 61.6, 92.4,
61.6, 163.4, 164.9; HRMS calculated for C H N O, 336.2889; found 336.2884.
19
36
4
2
4
N
-(3-Dimethylaminopropyl)-
N
-dodecyl-6-methylpyrimidine-2,4-diamine (8a)
ꢀdodecylꢀ6ꢀ
Synthesized using the general procedure as for 5a from 4ꢀchloroꢀ
N
methylpyrimidinꢀ2ꢀamine (4a) and N,Nꢀdimethylaminopropylamine. The residue was
purified by dry column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH
3
3
to yield the target compound.
8a) Yield 183 mg, 74% pale yellow oil; H NMR (CDCl
m, 18H), 1.46 (m, 2H), 1.63 (m, 2H), 2.05 (s, 3H), 2.11 (s, 6H), 2.24 (t, 2H,
3.14 (m, 2H), 3.29 (t, 4H, = 6.8 Hz), 4.18 (br s, 1H), 5.19 (br s, 1H), 5.45 (s, 1H); C NMR
1
(
3
): δ 0.78 (t, 3H,
J
= 6.5 Hz), 1.20
(
J = 6.7 Hz),
13
J
(CDCl ): δ 13.3, 21.9, 23.4, 26.3, 26.9, 28.6, 28.7 (2 x C), 28.9, 29.0 (2 x C), 29.1, 31.2, 39.2,
3
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
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5
5
5
5
5
6
43 5
40.6, 44.7, 56.9, 91.8 (br), 160.9, 163.1, 163.2 (br); HRMS calculated for C22H N ,
377.3518; found 377.3526.
4
2
N
-(3-Dimethylaminopropyl)-
Synthesized using the general procedure as for 5b from 2ꢀchloroꢀ
methylpyrimidinꢀ4ꢀamine (4b) and N,Nꢀdimethylaminopropylamine. The residue was
N
-dodecyl-6-methylpyrimidine-2,4-diamine (8b)
0
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2
3
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2
3
4
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0
N
ꢀdodecylꢀ6ꢀ
3 3
purified by dry column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH
to yield the target compound.
1
(8b) Yield 212 mg, 86% pale yellow oil; H NMR (CDCl
3
): δ 0.79 (t, 3H,
J
= 6.5 Hz), 1.19
= 6.8 Hz),
(
m, 18H), 1.45 (m, 2H), 1.63 (m, 2H), 2.02 (s, 3H), 2.12 (s, 6H), 2.25 (t, 2H,
J
13
3
.25 (m, 4H), 5.00 (br s, 1H), 5.45 (s, 1H), 5.52 (br s, 1H); C NMR (CDCl ): δ 13.3, 21.9,
3
23.2, 26.3, 26.4, 28.6, 28.7 (2 x C), 28.9 (2 x C), 29.0, 29.2, 31.2, 39.4, 41.4, 44.7, 57.2, 91.9
43 5
(br), 161.3, 163.1, 163.8 (br); HRMS calculated for C22H N , 377.3518; found 377.3529.
4
2
2
N
-Dodecyl-
Synthesized using the general procedure as for 5a from 4ꢀchloroꢀ
methylpyrimidinꢀ2ꢀamine (4a) and N,Nꢀdimethylamine. The residue was purified by dry
N ,N ,6-trimethylpyrimidine-2,4-diamine (9a)
N
ꢀdodecylꢀ6ꢀ
3 3
column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield the
target compound.
1
(
9a) Yield 34 mg, 14% pale yellow oil; H NMR (CDCl
3
): δ 0.85 (t, 3H,
J
= 6.9 Hz), 1.25
= 6.9 Hz), 4.88
(m, 18H), 1.54 (p, 2H,
J
= 7.4 Hz), 2.16 (s, 3H), 3.03 (s, 6H), 3.37 (q, 2H,
J
13
(
br s, 1H), 5.64 (s, 1H); C NMR (CDCl ): δ 13.5, 22.0, 24.2, 26.4, 28.7 (2 x C), 28.8 (2 x
3
C), 28.9, 29.0, 29.3, 31.3, 36.2, 40.8, 90.7, 161.4, 163.1, 164.7; Analysed for: C, 71.20; H,
11.32; Found C 71.15, H 11.21.
4
2
N
-(2-Dimethylaminoethyl)-
N
-dodecyl-6-methylpyrimidine-2,4-diamine (9b)
ꢀdodecylꢀ6ꢀ
Synthesized using the general procedure as for 5a from 2ꢀchloroꢀ
N
methylpyrimidinꢀ4ꢀamine (4b) and N,Nꢀdimethlamine. The residue was purified by dry
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3 3
column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield the
target compound.
1
(
9b) Yield 190 mg, 93%, mp 31ꢀ33°C; H NMR (CDCl
3
): δ 0.85 (t, 3H,
J = 6.33 Hz), 1.23
(m, 18H), 1.53 (m, 2H), 2.14 (s, 3H), 2.97 (s, 6H), 3.34 (q, 2H,
J
= 6.8 Hz), 4.79 (br s, 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
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51
52
53
54
55
56
57
58
59
60
1
3
5.61 (s, 1H); C NMR (CDCl ): δ 13.3, 22.0, 23.4, 26.3, 28.5, 28.6 (2 x C), 28.7, 28.8 (2 x
3
C), 28.9, 31.2, 38.3, 40.7, 90.6, 161.6, 163.1, 164.9; Analysed for: C, 71.20; H, 11.32; Found
C 70.82, H 11.03.
2
4
N
-(2-(Dimethylamino)ethyl)-6-methyl-
Synthesized using the general procedure as for 5a from 2ꢀchloroꢀ6ꢀmethylꢀ
propylpyrimidinꢀ4ꢀamine (10a) and N,N-dimethylethylenediamine. The residue was purified
N -propylpyrimidine-2,4-diamine (27a)
N
ꢀ
3 3
by dry column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield
the target compound.
1
(
27a) Yield 52 mg, 51% pale yellow oil; H NMR (CDCl
3
): δ 0.92 (t, 3H,
= 6.3 Hz), 3.19 (q, 2H, = 6.7 Hz), 3.40 (m,
H), 4.78 (br s, 1H), 5.21 (br s, 1H), 5.50 (s, 1H); C NMR (CDCl ): δ 10.8, 22.2, 23.1, 38.4,
J = 7.4 Hz), 1.56
(m, 2H), 2.14 (s, 3H), 2.20 (s, 6H), 2.43 (t, 2H,
J
J
1
3
2
4
3
21 5
2.5, 44.6, 58.0, 91.5 (br), 161.6, 163.3, 165.0; HRMS calculated for C11H N , 223.1797;
found 223.1790.
4
2
N
-(2-Dimethylaminoethyl)-6-methyl-
Synthesized using the general procedure as for 5b from 4ꢀchloroꢀ6ꢀmethylꢀ
propylpyrimidinꢀ2ꢀamine (10b) and N,N-dimethylethylenediamine. The residue was purified
N -propylpyrimidine-2,4-diamine (27b)
N
ꢀ
3 3
by short column chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield the
target compound.
1
(
27b) Yield 52 mg, 51% pale yellow oil; H NMR (CDCl
3
): δ 0.90 (t, 3H,
J = 7.4 Hz), 1.61
(
m, 2H), 2.19 (s, 6H), 2.21 (s, 3H), 2.36 (t, 2H,
J
= 6 Hz), 3.29 (m, 4H), 5.32 (br s, 1H), 5.50
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Journal of Medicinal Chemistry
Page 28 of 51
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
3
(s, 1H); C NMR (CDCl
3
): δ 10.8, 22.4, 34.9, 37.9, 42.5, 44.4, 57.3, 92.2 (br), 160.5, 161.1,
1
63.0; HRMS calculated for C11
H
21
N
5
, 223.1797; found 223.1806.
-(2-dimethylaminoethyl)-6-methylpyrimidine-2,4-diamine (28a)
Synthesized using the general procedure as for 5a from ꢀbutylꢀ2ꢀchloroꢀ6ꢀ
methylpyrimidinꢀ4ꢀamine (11a) and N,N-dimethylethylenediamine. The residue was purified
4
2
N
-Butyl-N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
3 3
by short column chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield the
target compound.
1
(28a) Yield 280 mg, 82% pale yellow oil; H NMR (CDCl
3
): δ 0.73 (t, 3H,
J
= 7.3 Hz),
= 6.5 Hz),
1
3
1
.18 (m, 2H), 1.33 (q, 2H,
.08 (q, 2H, = 6.8 Hz), 3.25 (t, 2H,
H); C NMR (CDCl ): δ 13.0, 19.3, 22.7, 35.2, 38.2, 40.1, 44.5, 57.8, 91.8 (br), 161.5,
, 237.1953; found 237.1959.
-(2-dimethylaminoethyl)-6-methylpyrimidine-2,4-diamine (28b)
Synthesized using the general procedure as for 5b from ꢀbutylꢀ4ꢀchloroꢀ6ꢀ
methylpyrimidinꢀ2ꢀamine (11b) and N,N-dimethylethylenediamine. The residue was purified
J
= 6.6 Hz), 1.96 (s, 3H), 2.04 (s, 6H), 2.27 (t, 2H,
J
J
J = 6.4 Hz), 4.13 (br s, 1H), 5.17 (br s, 1H), 5.38 (s,
1
3
3
23 5
163.1, 164.0 (br); HRMS calculated for C12H N
2
4
N
-Butyl-N
N
3 3
by dry column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield
the target compound.
1
(
28b) Yield 191 mg, 85% pale yellow oil; H NMR (CDCl
3
): δ 0.81 (t, 3H,
J
= 7.3 Hz),
1.25 (m, 2H), 1.41 (p, 2H,
J
= 6.7 Hz) 2.02 (s, 3H), 2.11 (s, 6H), 2.35 (t, 2H,
J
= 6.2 Hz),
13
3
.24 (m, 4H), 4.08 (br s, 1H), 5.06 (br s, 1H), 5.53 (s, 1H); C NMR (CDCl ): δ 13.1, 19.4,
3
2
2.7, 31.4, 37.8, 40.3, 44.5, 57.5, 92.1 (br), 161.5, 163.0, 163.9; HRMS calculated for
C
12
H
23
N
5
, 237.1953; found 237.1965.
4
2
N
-Butyl-
N
-(2-dimethylaminoethyl)-6-methylpyrimidine-2,4-diamine (29a)
ꢀbutylꢀ2ꢀchloroꢀ6ꢀ
Synthesized using the general procedure as for 5a from
N
methylpyrimidinꢀ4ꢀamine (12a) and N,N-dimethylethylenediamine. The residue was purified
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Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
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by dry column flash chromatography using EtOAc followed by 1:1 CHCl
3
:CH
3
OH to yield
the target compound.
1
(
29a) Yield 280 mg, 82% pale yellow oil; H NMR (CDCl
3
): δ 0.73 (t, 3H,
J
= 7.3 Hz),
= 6.5 Hz),
1.18 (m, 2H), 1.33 (q, 2H,
J
= 6.6 Hz), 1.96 (s, 3H), 2.04 (s, 6H), 2.27 (t, 2H,
J
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3.08 (q, 2H,
J
= 6.8 Hz), 3.25 (t, 2H, J = 6.4 Hz), 4.13 (br s, 1H), 5.17 (br s, 1H), 5.38 (s,
13
1
H); C NMR (CDCl
3
): δ 13.0, 19.3, 22.7, 35.2, 38.2, 40.1, 44.5, 57.8, 91.8 (br), 161.5,
, 251.2110; found 251.2119.
-(2-dimethylaminoethyl)-6-methylpyrimidine-2,4-diamine (29b)
Synthesized using the general procedure as for 5b from ꢀbutylꢀ4ꢀchloroꢀ6ꢀ
methylpyrimidinꢀ2ꢀamine (12b) and N,N-dimethylethylenediamine. The residue was purified
163.1, 164.0 (br); HRMS calculated for C13
25 5
H N
2
4
N
-Butyl-N
N
3 3
by dry column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield
the target compound.
1
(29b) Yield 191 mg, 85% pale yellow oil; H NMR (CDCl
3
): δ 0.81 (t, 3H,
J
= 7.3 Hz),
1
3
2
.25 (m, 2H), 1.41 (p, 2H,
J
= 6.7 Hz) 2.02 (s, 3H), 2.11 (s, 6H), 2.35 (t, 2H,
J
= 6.2 Hz),
13
.24 (m, 4H), 4.08 (br s, 1H), 5.06 (br s, 1H), 5.53 (s, 1H); C NMR (CDCl ): δ 13.1, 19.4,
3
2.7, 31.4, 37.8, 40.3, 44.5, 57.5, 92.1 (br), 161.5, 163.0, 163.9; HRMS calculated for
C
13
H
25
5
N , 251.2110; found 251.2120.
2
4
N
-(2-Dimethylaminoethyl)-
N
-hexyl-6-methylpyrimidine-2,4-diamine (30a)
ꢀhexylꢀ6ꢀ
methylpyrimidinꢀ4ꢀamine (13a) and N,N-dimethylethylenediamine. The residue was purified
Synthesized using the general procedure as for 5a from 2ꢀchloroꢀ
N
3 3
by dry column flash chromatography using EtOAc followed by 1:1 CHCl :CH OH to yield
the target compound.
1
(30a) Yield 36 mg, 15% pale yellow oil; H NMR (CDCl
3
): δ 0.88 (t, 3H,
J
= 7.3 Hz), 1.28
(m, 6H), 1.53 (m, 2H), 2.17 (s, 3H), 2.26 (s, 6H), 2.46 (t, 2H,
J
= 6.3 Hz), 3.20 (q, 2H, J = 5.9
13
Hz), 3.41 (m, 2H), 4.70 (br s, 1H), 5.30 (br s, 1H), 5.55 (s, 1H); C NMR (CDCl ): δ 13.3,
3
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