Synthesis, molecular modeling and biological activity of methyl and thiomethyl substituted pyrimidines as corticotropin releasing hormone type 1 antagonists

Article abstract of DOI:10.1039/b305458f

Four small, targeted libraries of differentially substituted amino pyrimidines were synthesized in moderate to good yields. Excellent regiochemistry was observed for substitution at C2/C4 with selectivity >50 : 1 noted. All analogues were screened for their ability to interact with CRH ₁ and CRH₂ receptors. In all instances only poor agonistic and/or antagonistic behaviour was noted at CRH₂. However, several compounds were potent and selective CRH₁ antagonists, most notably 13a K₁ = 39 nM. Additionally we have utilized these data and that recently reported by others to refine our original CRH₁ pharmacophore (J. Med. Chem., 1999, 42, 2351-2357).

Full text of DOI:10.1039/b305458f

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Synthesis, molecular modeling and biological activity of methyl
and thiomethyl substituted pyrimidines as corticotropin releasing
hormone type 1 antagonists†
Adam McCluskey,*a Paul A. Keller,*b Jody Morganb and James Garnera
a Chemistry, School of Environmental & Life Sciences, The University of Newcastle, Callaghan,
NSW 2308, Australia. E-mail: amcclusk@mail.newcastle.edu.au;
Fax: 61 24921 5472; Tel: 61 24921 6486
b Department of Chemistry, The University of Wollongong, Wollongong, NSW 2522, Australia.
E-mail: paul_keller@uow.edu.au; Fax: 612 4221 4287; Tel: 61 24221 4692
Received 15th May 2003, Accepted 6th August 2003
First published as an Advance Article on the web 22nd August 2003
Four small, targeted libraries of differentially substituted amino pyrimidines were synthesized in moderate to good
yields. Excellent regiochemistry was observed for substitution at C2/C4 with selectivity >50 : 1 noted. All analogues
were screened for their ability to interact with CRH1 and CRH2 receptors. In all instances only poor agonistic
and/or antagonistic behaviour was noted at CRH2. However, several compounds were potent and selective CRH1
antagonists, most notably 13a Ki = 39 nM. Additionally we have utilized these data and that recently reported by
others to refine our original CRH1 pharmacophore (J. Med. Chem., 1999, 42, 2351–2357).
receptor involved in human parturition is of the type 1.11
Introduction
Therefore we might reasonably be able to predict that a type 1
Corticotropin releasing hormone (CRH) is a 41 amino acid
receptor antagonist would inhibit the progression of labour
peptide implicated in a number of physiological conditions,
examples of which include (but are not limited to) anorexia
nervosa, Alzheimer’s disease, the mediation of stress responses,
and conversely, a type 1 receptor agonist would promote the
onset of labour. Recently Smith et al. demonstrated the use of
antalarmin (1) in delaying the onset of labour in a controlled
and being linked to a biological clock as the initiator of the
study involving pregnant ewes.12 We and others have recently
onset of labour.1 Our primary interest is in the interaction of
reviewed the therapeutic implications and medicinal chemistry
CRH and the onset of human parturition.
While the hormonal mechanisms that control the onset of
developments in corticotropin releasing hormone related
research.13
parturition and labour are largely unknown, a large body
Because of the key role of the CRH1 receptor in human par-
of data suggests that CRH plays a key role in this process.
turition, and the lack of an alternative suitable treatment, there
Placental CRH concentrations rise exponentially during preg-
nancy in maternal circulation. McLean et al. have demon-
is an urgent need for low molecular weight ligands that are
active at this receptor either as agonists or antagonists. Such
ligands would not only serve as potential therapeutics, but
strated that the exponential rise in CRH is linked to a biological
clock which determines the length of gestation.2 Various groups
could lead to a greater understanding of the structure and func-
have shown that women in preterm labour have higher CRH
tion of the receptor itself. There are numerous peptidic and
concentrations than gestationally matched controls.3,4 Smith’s
non-peptidic antagonists and agonists currently available, but
group has also demonstrated similar results from different per-
the drive remains to produce a therapeutically acceptable sub-
spectives.5–8 Firstly, women undergoing induction of labour are
stance, in combination with the apparent limited structural
more likely to have a successful induction if CRH concen-
diversity of these known compounds.10,14–18
trations are high. Secondly, women who are in apparent pre-
Over the past 5–10 years there has been an up-surge in the
term labour are significantly more likely to deliver prematurely
if they have high CRH levels.
number, but unfortunately, not the diversity in the types of
small molecules shown to interact specifically with CRH1 recep-
tors. However, such a situation can be advantageous in pro-
ducing a detailed structure–activity relationship (SAR) study,
and thus, we developed a pharmacophore for this receptor.12
With this in mind, we have revisited the substituted pyrimidines
developed by Whitten et al.19 in producing additional analogues
for investigations into pharmacophore development.20 Herein
we wish to report upon our recent results, in the development of
CRH1 antagonists and pharmacophore refinement.
Chemistry
In our efforts to further refine our published pharmacophore
we sought simple and expedient routes to the synthesis of small
targeted libraries of differentially substituted pyrimidines. We
envisaged the development of four such libraries, exemplified
by type A–D compounds shown in Fig. 1.
CRH receptors are members of the 7-transmembrane family
and are normally linked via a G-protein to adenylate cyclase.9
Several types of receptor have been described,10 CRH1, CRH2α,
CRH2β and more recently, CRH2ψ, although the key CRH
Type A analogues most closely resemble those reported by
Whitten et al., however types B–D allow for subtle changes in
the spatial orientation of key substituents which should be
† Electronic supplementary information (ESI) available: detailed
description of pharmacophore development using CATALYST©. See
http://www.rsc.org/suppdata/ob/b3/b305458f/
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Fig. 1 Targeted libraries of differentially substituted pyrimidines as potential CRH antagonists for pharmacophore refinement.
Scheme 1 Synthesis of type A and B compounds (i) POCl3 (15 equiv.), reflux 5 h; (ii) option ‘a’ NaH–THF, TCA, 17–24 h; (ii) option ‘b’ 5 M
LPDE–amine, 12 h; (iii) amine, sealed tube, 160 ЊC.
reflected in differing activities at CRH1 receptors, and con-
sequently allow further refinement of our pharmacophore.20
Previously efforts from our laboratory have shown that it is
possible to selectively substitute at either the 4- or 6-chloro
substituent in a range of 4,6-dichloropyrimidines where the
C2 position is occupied by either a methyl or a thiomethyl moi-
ety.21 In order to use our in-house methodology we first syn-
thesized the appropriate dichloropyrimidines, thus treatment of
the parent dihydroxypyrimidines (2–5) with an excess of POCl3
at reflux affords, cleanly, the desired dichloro species (6–9) in
good to excellent yields (Schemes 1 and 2, Table 1 entries 1, 9,
17, 28).
We had originally envisaged that subsequent displacement of
the thiomethyl moiety in 7, would afford rapid access to
appropriately substituted dichloroamino pyrimidines. Amino-
lysis reactions of this nature are known to be typical in
pyrimidine chemistry. Subsequent efforts using typical condi-
tions: sealed tube, 110 ЊC, 24–72 h, EtOH, NHR2, as previously
described by Chebib et al.22 afforded none of the anticipated
thiomethyl substitution product. In all instances, we detected
products corresponding to nucleophilic substitution at C2
(minor isomer) and C4 (major isomer) (data not shown).
The selective displacement of the C4 chlorine led us to
explore the possible use of alternate reaction media in an
attempt to improve our observed selectivity. Accordingly, we
examined the use of 5 M lithium perchlorate–diethyl ether
(LPDE) solutions as a solvent system for the selective reaction
at C4 in 6 and 7; and at C2 and/or C6 in 8 and 9. Gratifyingly
good selectivity (>20 : 1) was observed, especially with sym-
metrically substituted dichloropyrimidines 6 and 7 (option ‘a’
in Schemes 1 and 2). Typically, a symmetrically substituted
dichloropyrimidine stirred overnight at room temperature in
scrupulously anhydrous LPDE along with an amine, followed
by extractive work-up gave, in all instances, mono-substituted
products in moderate to good yields ( Table 1, entries: 3–5, 11–
13). The second chlorine was then readily substituted with a
range of amines by simply heating the amine and pyrimidine in
a sealed tube at 160 ЊC, with moderate yields for type A or B
compounds. A slight reduction in selectivity was observed with
4 and 5, although in these latter cases one isomer dominated.
Indeed, in some instances, the second isomer could not be
detected.
Subsequent stepwise amination of 6 and 7 in the C4 position,
followed by the C6 position, yielded two series of 2-methyl-4,6-
diaminated pyrimidines (12 and 13) (Scheme 1); and amination
of 8 and 9 in the equivalent C2 and C4 positions yields a series
of 5/6-methyl-2,4-diaminated pyrimidines (18 to 21) (Scheme
2).
The synthesis of the C5 and C6 methylated pyrimidines, type
C and D compounds, was undertaken using typical pyrim-
idine chemistry by amination of the corresponding dichloro
derivatives (option ‘a’ in Schemes 1 and 2). Highlighting the
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Scheme 2 Synthesis of type C and D compounds. (i) POCl3 (15 equiv.), reflux 5 h; (ii) option ‘a’ NaH–THF, TCA, 17–24 h; (ii) option ‘b’ 5 M
LPDE–amine, 12 h; (iii) amine, sealed tube, 160 ЊC.
effectiveness of selectivity shown by the synthesis for the type A
and B compounds, these compounds showed little selectivity in
amination and required chromatographic separation of the
regioisomers.
combination affords greater water solubility, a considerable
advantage in this field. Given the unexplored nature of this
small binding pocket and the restriction in total steric bulk, we
felt that these data warranted a reexamination of our prelimin-
ary pharmacophore.20
The activities of these pyrimidine analogues against the CRH
type 1 and 2 receptors are shown in Table 1. A negative value
for CRH2 indicates agonist activity whereas a positive value
shows an antagonist activity. A dash indicates no clear outcome
was resolved. All CRH2 data, expressed as a %-inhibition of the
suavagine mediated stimulation of cAMP, was obtained at 100
µM drug concentration. In our laboratory <40% inhibition at
100 µM is operationally defined as inactive and not worthy of
further examination, hence none of the analogues shown in
Table 1 showed sufficient promise to warrant Ki determination.
On examining the results for effectiveness against the CRH1
receptor, it is clear the presence of a chloro substituent (6–9)
attached directly to the pyrimidine ring shows no activity.
Somewhat disappointingly, neither type C nor type D com-
pounds showed any activity against CRH1 ( Table 1, entries
17–36; Ki > 2000 nM). With type A and B compounds,
similar results were noted for the simple mono-aminated ana-
logues (Table 1, entries 1–5 and 9–13; Ki > 2000 nM). This is in
keeping with previous reports.19 The introduction of a second
amino group into type A compounds afforded the first indi-
cation of biological activity against CRH1 (Table 1, entry 6, R5
= nPrNH; Ki = 1429 nM). Increasing the bulk of this substituent
significantly improves activity (Table 1, entries 7 and 8, R5 =
iBuNH and nPrNCHcPr; Ki = 986 and 130 nM respectively).
These data suggest that the presence of a bulky hydrophobic
substituent distal to the TCA moiety increases activity, indeed
the tertiary amine (12c) was the most active of the type A
compounds examined. However, examination of type B com-
pounds showed the opposite trend, that is a reduction in CRH1
potency as a function of R5 bulk ( Table 1, entries 14–16; Ki =
39, 376 and >2000 nM respectively). This modification in
potency must be a function of the thiomethyl moiety, however
it is not apparent whether this is due to increased steric bulk or
the introduction of the sulfur lone pairs, or a combination of
both. These data do, however, suggest that there remains a
small binding pocket, as yet largely unexplored. Additionally,
these data suggest that the introduction of a thiomethyl moiety
necessitates a reduction in steric bulk in the R5 substituent. This
Pharmacophore development for the CRH1 receptor
Since our 1st generation pharmacophore model of the CRH1
receptor,20 numerous publications have appeared containing
novel antagonists of this receptor.13,14 In an iterative process, we
have refined our original pharmacophore to include these
compounds and have also investigated the effects of the novel
thiomethyl derivatives from this paper on these models. All
pharmacophore modeling work has been done using CATA-
LYST© 1991–1999 (for a more detailed description of pharma-
cophore development using CATALYST©, see the attached
electronic supplementary information† and references cited
therein).23–25 Briefly, the refinement undertaken in this study
involved, the inclusion of novel recently reported CRH1
antagonists, the removal of redundant data and ambiguous
data. Further refinement involved the inclusion of activity data
spanning four orders of magnitude (nM–µM), avoiding bias
towards highly active analogues. This 2nd generation CRH1
receptor pharmacophore is shown in Fig. 2.
The 2nd generation hypothesis (Fig. 2) has a null Ϫ total cost
difference of 144 bits suggesting this model has a greater than
90% probability of being a true representation of the data (refer
to Table 2 for cost values). The model contained one hydrogen
bond acceptor, one ring aromatic and three hydrophobic
regions and accurately predicted the activity of a test set of data
(correlation 0.989).26
The 3rd generation model (Fig. 3) involved the additional
inclusion of the data from this paper into the previous training
set, in particular the inclusion of compounds showing higher
activity when containing the thiomethyl group (13a and 13b). It
was believed that this feature may be highlighted as being
important for activity by the modeling program. However, this
proved to not be the case with the addition of these compounds
having very little effect on the appearance of the pharmaco-
phore, containing the same features as the previous model
(Fig. 2). It did, however, cause a change in the cost of the
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Table 1 Antagonist activity against CRH type 1 and type 2 receptors
Entry
Compound
R groups
CRH1 Ki/nM
CRH2 (%)a
Yield (%)
79
Type A compounds
1
6
>2000
3
R3
R4
2
3
4
5
10a
10b
10c
10d
CH3
CH3
CH3
CH3
TCA
>2000
>2000
>2000
>2000
Ϫ5
Ϫ3
17
82
57
38
25
nPrNH
iBuNH
nPrN-CH2cPr
—
R5
6
7
8
12a
12b
12c
CH3
CH3
CH3
TCA
TCA
TCA
nPrNH
iBuNH
nPrN-CH2cPr
1429
986
130
13
16
12
47
34
79
Type B compounds
9
7
>2000
5
88
R3
R4
10
11
12
13
11a
11b
11c
11d
SCH3
SCH3
SCH3
SCH3
TCA
>2000
>2000
>2000
>2000
—
6
6
60
61
61
27
nPrNH
iBuNH
nPrN-CH2cPr
13
R5
14
15
16
13a
13b
13c
SCH3
SCH3
SCH3
TCA
TCA
TCA
nPrNH
iBuNH
nPrN–CH2cPr
39
376
>2000
24
Ϫ2
Ϫ2
55
78
47
Type C compounds
17
8
H
>2000
13
64
R4
TCA
nPrNH
iBuNH
Pr-N-CH2cPr
—
—
—
R5
R6
H
H
H
H
H
H
H
H
H
H
R7
18
19
20
21
22
23
24
25
26
27
14a
14b
14c
14d
16a
16b
16c
16d
18a
20a
—
—
—
—
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
>2000
13
13
8
Ϫ10
8
—
17
13
—
56
7
41
12
29
50
23
46
53
27
TCA
nPrNH
iBuNH
Pr-N-CH2cPr
Pr-N-CH2cPr
TCA
—
TCA
Pr-N-CH2cPr
—
Type D compounds
28
9
CH3
R6
H
R7
H
H
H
H
H
H
H
H
>2000
>2000
Ϫ33
Ϫ39
72
4b
R4
R5
—
—
—
29
30
31
32
33
34
35
36
TCA
nPrNH
iBuNH
nPrN-CH2cPr
—
—
—
—
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
c
—
>2000
—
>2000
>2000
>2000
>2000
Ϫ10
Ϫ30
Ϫ38
Ϫ28
Ϫ8
1.6b
0.15b
68b
—
TCA
nPrNH
iBuNH
nPrN-CH2cPr
46b
43b
Ϫ42
43b
a % Inhibition of the sauvagine-stimulated adenosine 3Ј,5Ј-phosphate (cAMP) at a drug concentration of 100 µM. b Selectivity 17a : 15a = 17 : 1;
17b : 15b = >50 : 1; 17c : 15c = 27 : 1; 17d : 15d = >50 : 1. c None detected in the reaction mixture.
Table 2 Cost values for the generation of all three pharmacophore models, where the fixed cost (cost of a perfect hypothesis), the null cost (cost of a
hypothesis for which we assume there is no structural data) and the total cost (the cost for the hypothesis generation) are all measured in arbitrary
units (bits)
2nd generation model
3rd generation model
Fixed cost
Null cost
Total cost
Null Ϫ total cost difference
433.85
797.18
653.52
143.66
474.05
885.25
713.85
171.4
hypothesis model, significantly increasing the null Ϫ total cost
difference and therefore increasing the statistical validity of the
pharmacophore model. This also resulted in the highest scoring
(lowest cost) hypothesis being selected as the best hypothesis
model. This unexpected occurrence leads to this ‘third gener-
ation model’ being the most statistically valid and therefore
presumably the most accurate model to date.
In order to test the possibility that the range of derivatives
containing the thiomethyl substituents provided sufficient
structure–activity information to generate a valid pharmaco-
phore of equal quality to either/both our 2nd generation or 3rd
generation models, a separate model was created using only
compounds reported in this paper. One important question to
ask was, given that the training set would contain a higher
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Fig. 4 Hypotheses from the thiomethyl model, generated using only
Fig. 2 The highest scoring hypothesis for the ‘second generation
model’ generated by CATALYST. (a) Overlay of 13a on this hypothesis,
estimated activity of 2.7 nM (observed Ki = 39 nM). (b) Cartoon
representation of 13a mapped onto the hypothesis highlighting actual
groups and atoms that correspond with the CATALYST image in panel
(a); the regions of importance in the molecule: green: hydrogen bond
acceptor, orange: ring aromatic, blue: hydrophobic aliphatic.
compounds from this paper. The highest scoring hypothesis overlayed
on 13a does not map the thiomethyl group as an important feature,
estimate activity of 26 nM (observed Ki = 39 nM).
pharmacophore without apparently being directly related to a
proposed ‘receptor’ interaction. Rather, the presence of these
substituents contributes to the electronic nature of the SAR, or
the conformational analysis of the ligands giving rise to the
pharmacophore model to yield a more accurate representation,
both electronically and spatially, of the proposed binding site
shape of the CRH1 receptor antagonists. In these studies, how-
ever, the true importance of the thiomethyl moiety may have
been concealed by the large size of the training set with so
few compounds containing this functional group. We are cur-
rently aiming towards generating a larger array of thiomethyl
based compounds for screening and incorporation into our
pharmacophore model. These models are currently being
utilized in further studies, both in database mining and de novo
design experiments in an attempt to generate novel, structurally
variable antagonists of CRH1.
Experimental
General methods
Fig. 3 (a) The highest scoring hypothesis for the ‘generation three
model’ overlayed with 13a, estimated activity of 2 nM (observed Ki =
39 nM). (b) An overlay of the ‘generation two’ and ‘generation three’
models showing their similarity.
THF and ether were freshly distilled from sodium–benzo-
phenone. Flash chromatography was carried out using silica gel
200–400 mesh (60 Å). H and 13C NMR were recorded at 300
1
MHz and 75 MHz respectively using a Bruker Avance 300 MHz
spectrometer in CDCl3.
proportion of derivatives with a thiomethyl substituent, would
this feature show as being an important feature in the final
model?
GCMS was performed using a Shimadzu GCMS-QP5050A.
The instrument uses a quadrupole mass spectrometer and
detects samples via electron impact ionization (EI). The
University of Wollongong Biomolecular Mass Spectrometry
Laboratory analysed samples for HRMS. The spectra were run
on the VG Autospec-oa-tof tandem high resolution mass
spectrometer using CI ϩve (chemical ionization), with methane
as the carrier gas and PFK (perfluorokerosene) as the reference.
The training set for this model was small, containing only 13
compounds, and therefore was not expected to yield a statistic-
ally valid result. The returned highest scoring hypothesis is
shown in Fig. 4, with compound 13a overlaid. The highest
scoring hypothesis contained 2 hydrophobic features, 1 ring
aromatic and 1 hydrogen bond acceptor feature; none of these
features however, mapped the thiomethyl substituent (Fig. 4).
Therefore, even though the model possesses a low statistical
validity (indicating that it should be treated with caution), and
despite a heavy weighting in the training set of molecules con-
taining a thiomethyl substituent, the result lends support to the
conclusion that the improvement in our 3rd generation model is
valid and the lack of the thiomethyl overlaying on top of a
‘feature’ of the pharmacophore is more than likely a true
representation.
In conclusion, the inclusion of the thiomethyl containing
compounds from this paper led to an increase in the statistical
validity of the current pharmacophore model for the CRH1
receptor without selecting this group as being important for
activity. The significance of this analysis lies in the continual
refinement of the model, with the SAR consideration that
inclusion of specific substituents can increase the quality of the
Stimulation of cAMP production by sauvagine
Adenylate cyclase activity was performed using cells transfected
with either the human CRFl or CRF2α receptor. Cells were
plated onto 24-well cell culture plates and grown to confluency.
On the day of assay, the plates were removed from the incu-
bator, the medium was aspirated, and the cells were washed
once with PBS (phosphate buffered saline). Assays were carried
out at 37 ЊC for 1 h in a final volume of 0.5 ml in assay buffer
containing Dulbecco’s modified Eagle’s medium, 2 mM
-glutamine, 20 mM HEPES, and 1 mM 3-isobutyl-1-methyl-
xanthine. In stimulation studies, various concentrations of CRF-
related and -unrelated peptides were incubated with the cells to
establish the pharmacological rankorder profile of this receptor
subtype. For the functional assessment of antagonists [α-helical
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CRF(9–41) or -Phe-CRF(12–41)], 10 nM sauvagine (causing
∼60–80% stimulation of cAMP production) was incubated
along with various concentrations of competing peptides (10Ϫ12
to 10Ϫ6 M). After the incubations, the medium was aspirated,
the wells were rinsed once gently with fresh medium and aspir-
ated. Intracellular cAMP was extracted from the cells by the
addition of 300 µ1 of a solution of 95% ethanol containing
20 mM HCl and incubated at Ϫ20 ЊC for 16–18 h. The solution
was then transferred to 1.5-ml Eppendorf tubes, and the wells
were washed with an additional 200 µ1 of 95% ethanol contain-
ing 20 mM HCI and pooled with the first fraction. The samples
were lyophilized and resuspended with 500 µ1 of sodium
acetate buffer. The measurement of cAMP in the samples was
performed using a single antibody cAMP radioimmunoassay
kit (Biomedical Technologies, Stoughton, MA).
at equilibrium at 22 ЊC as determined by the association
experiments described above. Specific binding was determined
at each concentration of radioligand in the presence of 1 µM
-Phe-CRF(12–41). This concentration was chosen from direct
competition studies demonstrating that at a concentration of
1 µM, -Phe-CRF could displace >95% of 125I-Tyr0-sauvagine
binding from CHO cells expressing the CRF2α, receptor.
Data analysis
All data analyses, including Scatchard, competition and kinetic
experiments, were analysed using the iterative nonlinear least-
squares curve-fitting program Prism (GraphPAD, San Diego,
CA). The saturation analyses of 125I-Tyr0-sauvagine binding
yielded Kd values that were subsequently used in the calculation
of the apparent Ki values for competing ligands in competition
assays performed under identical conditions. For competition
curves, the data were routinely fit to single- and multiple-site
models, and the fits were compared statistically to determine
whether a more-complex data model was justified.
125I-tyr0-sauvagine binding studies
Radioligand binding of 125I-Tyr0-sauvagine in transfected CHO
cells was performed essentially as previously described in detail
for the binding of either 125I-r/hCRF or 125I-oCRF to many
different tissues. All drugs and reagents (i.e., guanine nucleo-
tides) were made up in assay buffer, which was the same as the
tissue buffer described above with the addition of 0.15 mM
bacitracin, pH 7.0, at 22 ЊC. Briefly, 1.5 ml Eppendorf tubes
received (in order) 100 µ1 of buffer (with or without competing
peptides, guanine nucleotides, and so on), 50 µl of 125I-Tyr0-
sauvagine, and 150 µl of the membrane suspension as described
above, for a total assay volume of 300 µl. The assay was incu-
bated at equilibrium routinely for 2 h at 22 ЊC as determined by
direct kinetic experiments described below. Non-specific bind-
ing was determined in the presence of 1 µM unlabeled peptide
antagonist -Phe-CRF. Reactions were terminated by centri-
fugation in a Beckman microfuge for 10 min at 12000 × g. The
resulting pellets were washed gently with 1 ml of ice-cold PBS
containing 0.01% Triton X-100 and centrifuged again for
10 min at 12000 × g. The supernatants were aspirated, and the
tubes were cut just above the pellet, placed into 12 × 75 mm
polystyrene tubes, and monitored for radioactivity in a Packard
Cobra II γ-counter at ∼80% efficiency.
Kinetic studies were performed to determine the association
and dissociation rate constants for 125I-Tyr0-sauvagine and
determine the optimal time for equilibrium binding. Associ-
ation studies were initiated with the addition of the membrane
suspension to triplicate tubes containing the radiolabel (100–
200 pM final concentration) and buffer, with or without 1 µM
-Phe-CRF to define nonspecific binding. Tubes were allowed
to incubate for various times before centrifugation and washing
of the membranes. Specific binding was calculated for each time
point, and the rate constant for the association was determined
For dissociation experiments, tubes were set up as described for
the association assays and allowed to incubate for 2 h at 22 ЊC
After the 2 h incubation, 1 µM -Phe-CRF (final concen-
tration) was added to all tubes (total and nonspecific) to initiate
dissociation of the label in a final volume of 10 µl to minimize
the effects of dilution. The tubes again were centrifuged at
various times, and the specific binding was used to calculate the
dissociation rate constant as described below.
Synthesis
4,6-Dichloro-2-methylpyrimidine
(6).
4,6-Dihydroxy-2-
methylpyrimidine (5.0 g, 40 mmol) was mixed with POCl3
(30 mL, 15 eq) and refluxed for three hours, cooling to room
temperature, the reaction mixture was added dropwise to
vigorously stirred ice water (200 mL) in a salt–ice bath. The
yellow solid formed was collected by vacuum filtration (2.82 g).
The filtrate was extracted with ethyl acetate (2 × 70 mL). The
extract was washed with brine (30 mL), dried over MgSO4 and
evaporated in vacuo to give an additional 0.86 g of light brown
solid. Recrystallization with ethyl acetate gave a light brown
solid in a total yield of 3.70 g, 58%.
Mp 48–49 ЊC, 1H NMR: 2.75 (s, 3H), 7.25 (s, 1H); 13C NMR:
26.25, 118.97, 162.17, 170.39. GCMS: 163.0; calcd 163.0.
4-Chloro-2-methyl-6-(2Ј,4Ј,6Ј-trichloroanilino)pyrimidine
(10a). A solution of 2,4,6-trichloroaniline (400 mg, 2.0 mmol)
in THF (8 mL) was treated with NaH (160 mg, 60% dispersion
in oil, 4.0 mmol) at room temperature, under nitrogen. The
yellow suspension was stirred at room temperature for 10 min-
utes before 4,6-dichloro-2-methylpyrimidine (6) (0.326 mg,
2.0 mmol) was added and the mixture heated at reflux under
nitrogen for 24 hours. The reaction was quenched with ice water
(5 mL) and the product was extracted with ethyl acetate (3 ×
30 mL). The combined organic extracts were washed with brine
(25 mL), dried (MgSO4), filtrated and the solvent removed
in vacuo to give a brown solid. Recrystallization from ethyl
acetate–hexanes gave a pale yellow solid, yield 0.530 g, 82%.
Mp 166–168 ЊC, 1H NMR: 2.42 (s, 3H), 5.93 (s, 1H), 7.46 (s,
2H), 7.67 (br s, 1H); 13C NMR: 26.16, 100.51, 129.72, 131.64,
134.94, 135.85, 161.30, 162.60, 169.13.
4-Chloro-2-methyl-6-(N-propylamino)pyrimidine (10b). 4,6-
Dichloro-2-methylpyrimidine (10a) (250 mg, 1.5 mmol) was
dissolved in 5 mL of 5 M LPDE. Propylamine (1.26 mL, 10 eq)
was added and the sealed container was stirred for 24 hours.
Water (10 mL) was added and the solution was extracted with
ether (3 × 30 mL). The combined organic layers were dried
(MgSO4) and the solvent removed in vacuo. The yellow solid
obtained was purified by flash chromatography using 15 : 85
ethyl acetate–hexanes yielding 10b as an off-white solid, yield
160 mg, 57%.
For competition studies, tubes received (in order) 50 µ1 of
buffer, 50 µl of competing peptides (final concentration, 1 pM
to 1 µM), 50 µ1 of 125I-Tyr0-sauvagine (final concentration,
100–200 pM), and 150 µl of membrane suspension as described
above. Homogenates were typically allowed to incubate for 2 h
at 22 ЊC, and the reaction was terminated by separation of the
bound from free radioligand by centrifugation as described
above.
For Scatchard saturation studies, tubes received (in order)
50 µl of buffer, 50 µl of 125I-Tyr0-sauvagine (final concentration,
10 pM to 2 nM), and 50 µl of buffer with or without -Phe-
CRF to define non-specific binding. All assays were carried out
1
Mp 69–70 ЊC, H NMR: 0.82 (t, 3H), 1.48 (q, 2H), 2.31 (s,
3H), 3.08 (br s, 2H, -CH2-NH-), 5.72 (br s, 1H), 6.10 (s, 1H); 13
C
NMR: 11.79, 22.77, 26.06, 43.69, 97.92, 159.95, 164.02, 168.42.
HRMS: (M ϩ H)ϩ = 186.07979; calcd, 186.07987 (35Cl).
4-Chloro-6-(N-isobutylamino)-2-methylpyrimidine (10c). Syn-
thesized using the general procedure as for (10b). The yellow
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 5 3 – 3 3 6 1
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solid obtained was purified by flash chromatography using 1 : 4
ethyl acetate–hexanes and gave the product as a yellow oil, yield
116 mg, 38%.
4-Chloro-6-(N-propylamino)-2-methylmercaptopyrimidine
(11b). Synthesized using the general procedure as for (10b). The
pale yellow solid was recrystallized in hexane to give white
crystals, yield 336 mg, 61%.
1H NMR: 0.84 (d, 6H), 1.76 (sept, 1H), 2.34 (s, 3H), 2.95
(br s, 2H, -CH2-NH-), 5.64 (br s, 1H), 6.08 (s, 1H); 13C
NMR: 20.61, 26.06, 28.63, 97.81, 160, 164.17, 168.40. HRMS:
(M ϩ H)ϩ = 200.09522; calcd, 200.09552 (35Cl).
Mp 115–116 ЊC, 1H NMR: 0.94 (t, 3H), 1.60 (sext, 2H), 2.46
(s, 3H), 3.21 (br s, 2H, -CH2-NH-), 5.17 (br s, 1H), 5.99 (s, 1H);
13C NMR: 11.92, 14.58, 22.96, 43.77, 96.31, 99.79, 163.33,
172.51. HRMS: (M ϩ H)ϩ = 218.05182; calcd, 218.05194 (35Cl).
4-Chloro-6-(N-propyl-N-cyclopropylmethylamino)-2-methyl-
pyrimidine (10d). Synthesized using the general procedure as for
(10b). The brown solid obtained was purified by flash chrom-
atography using 1 : 4 ethyl acetate–hexanes and gave the product
as a yellow oil; yield 36 mg, 25%.
4-Chloro-6-(N-isobutylamino)-2-methylmercaptopyrimidine
(11c). Synthesized using the general procedure as for (10b). The
yellow solid was purified by flash chromatography using 15 : 85
ethyl acetate–hexanes leaving the product as a cream solid, yield
181 mg, 61%.
1H NMR: 0.17 (dd, 2H), 0.40 (dd, 2H), 0.80 (t, 3H), 0.90 (m,
1H), 1.5 (sext, 2H), 2.3 (s, 3H), 3.23 (br s, 2H ϩ 2H), 6.1 (s, 1H);
13C NMR: 4.17, 10.02, 11.77, 20.92, 26.40, 50.46, 52.70, 98.32,
159.74, 162.87, 168.03. HRMS: (M ϩ H)ϩ = 240.12657; calcd,
240.12682 (35Cl).
Mp 72–75 ЊC, 1H NMR: 0.89 (d, 6H), 1.82 (non, 1H), 2.43 (s,
3H), 3.05 (br s, 2H, -CH2-NH-), 5.42 (br s, 1H), 5.99 (s, 1H); 13
C
NMR: 14.59, 20.70, 28.81, 49.55, 96.25, 99.75, 163.48, 192.51.
HRMS: (M ϩ H)ϩ = 232.06722; calcd, 232.06757 (35Cl).
4-Chloro-6-(N-propyl-N-cyclopropylmethylamino)-2-methyl-
mercaptopyrimidine (11d). Synthesized using the general
procedure as for (10b). The brown solid was purified by flash
chromatography using 1 : 4 ethyl acetate–hexanes to give a
yellow oil, yield 38 mg, 27%.
2-Methyl-4-(N-propylamino)-6-(2Ј,4Ј,6Ј-trichloroanilino)-
pyrimidine (12a). A solution of 4-chloro-2-methyl-6-(2Ј,4Ј,6Ј-
trichloroanilino)pyrimidine (10a) (150 mg, 0.46 mmol) in dry
THF (10 mL) was added to a pressure vessel with propylamine
(0.39 mL, 10 eq). The vessel was flushed with nitrogen, sealed
and stirred at 160 oC for 24 hours. The solvent was removed in
vacuo to give a brown solid which was purified by flash chroma-
tography using 1 : 4 ethyl acetate–hexanes yielding a pale yellow
solid, yield 75 mg, 47%.
1H NMR: 0.18 (dd, 2H), 0.42 (dd, 2H), 0.81 (t, 3H), 0.93 (m,
1H), 1.52 (sext, 2H), 2.35 (s, 1H), 3.30 (br s, 2H ϩ 2H), 6.00 (s,
1H); 13C NMR: 4.11, 9.90, 11.69, 14.38, 20.93, 50.62, 52.86,
96.75, 159.34, 161.93, 171.79. HRMS: (M ϩ H)ϩ = 272.09861;
calcd, 272.09889 (35Cl).
Mp 156–158 ЊC, 1H NMR: 0.85 (t, 3H), 1.48 (sext, 2H), 1.95
(q, 2H), 2.12 (s, 3H), 4.73 (s, 1H), 5.29 (br s, 1H), 7.38 (s, 2H);
13C NMR: 12.06, 22.95, 25.73, 43.90, 78.75, 129.33, 133.44,
2-Methylmercapto-4-(N-propylamino)-6-(2Ј,4Ј,6Ј-trichloro-
anilino)pyrimidine (13a). Synthesized using the general pro-
cedure as for (12a). The brown solid product was purified by
flash chromatography using 1 : 4 ethyl acetate–hexanes to give a
yellow solid, yield 105 mg, 55%.
133.57, 136.02, 161.97, 164.07, 167.12. HRMS: (M ϩ H)ϩ
=
345.04272; calcd, 345.04413 (35Cl).
2-Methyl-4-(N-isobutylamino)-6-(2Ј,4Ј,6Ј-trichloroanilino)-
Mp 119–121ЊC, 1H NMR: 0.90 (t, 3H), 1.51 (sext, 2H), 2.93
(s, 3H), 3.07 (q, 2H), 4.79 (s, 1H), 4.85 (br s, 1H), 6.58 (br s, 1H),
7.38 (s, 2H); 13C NMR: 12.06, 14.40, 23.19, 43.93, 79.02,
129.27, 133.22, 133.39, 135.35, 161.02, 163.79, 171.24. HRMS:
(M ϩ H)ϩ = 377.01395; calcd, 377.01620 (35Cl).
pyrimidine (12b). Synthesized using the general procedure as for
(12a). The brown solid product was purified by flash chromato-
graphy using 1 : 4 ethyl acetate–hexanes to give a cream solid,
yield 56 mg, 34%.
1
Mp 159–162 ЊC, H NMR: 0.887 (d, 6H), 1.76 (sept, 1H),
2.23 (s, 3H), 2.84 (t, 2H), 4.78 (s, 1H), 5.11 (br s, 1H), 7.42 (s,
2H); 13C NMR: 20.20, 25.10, 28.16, 49.17, 78.40, 128.73,
132.59, 132.84, 134.98. 160.99, 163.20, 166.41.
2-Methylmercapto-4-(N-isobutylamino)-6-(2Ј,4Ј,6Ј-trichloro-
anilino)pyrimidine (13b). Synthesized using the general pro-
cedure as for (12a). The brown product was purified by flash
chromatography using 1 : 4 ethyl acetate–hexanes to give a
yellow solid, yield 129 mg, 78%.
2-Methyl-4-(N-propyl-N-cyclopropylmethylamino)-6-(2Ј,4Ј,6Ј-
trichloroanilino)pyrimidine (12c). Synthesized using the general
procedure as for (12a). The brown oil product was purified by
flash chromatography using 1 : 4 ethyl acetate–hexanes to give
an orange solid, yield 97.3 mg, 79%.
1
Mp 144–146 ЊC, H NMR: 0.87 (d, 6H), 1.78 (m, 1H), 2.94
(br t, 2H), 4.80 (br s, 1Hϩ1H), 4.83 (s, 3H), 6.34 (br s, 1H, NH),
7.40 (s, 2H); 13C NMR: 14.47, 20.93, 29.04, 49.80, 79.18,
129.33, 133.25, 133.36, 135.24, 160.96, 163.94, 171.33. HRMS:
(M ϩ H)ϩ = 391.03025; calcd, 391.03185 (35Cl).
Mp 126–128 ЊC, 1H NMR: 0.10 (dt, 2H), 0.29 (dt, 2H), 0.64
(t, 3H), 0.79 (m, 1H), 1.38 (sext, 2H), 2.23 (s, 3H), 3.25 (br d,
2H ϩ 2H), 4.87 (s, 1H), 7.40 (s, 2H); 13C NMR: 4.37, 10.47,
12.05, 21.30, 26.44, 50.43, 52.58, 77.28, 77.71, 78.13, 79.88,
129.30, 132.96, 134.03, 135.48, 161.26, 163.20, 167.12.
2-Methylmercapto-4-(N-propyl-N-cyclopropylmethylamino)-
6-(2Ј,4Ј,6Ј-trichloroanilino)pyrimidine (13c). Synthesized using
the general procedure as for (12a). The brown oil was purified
by speedy column using 1 : 4 ethyl acetate–hexanes to give a
white crystalline solid, yield 115 mg, 47%.
4,6-Dichloro-2-methylmercaptopyrimidine (7). Synthesized
using the general procedure as for (6). Recrystallization with
MeOH yielded a white solid, yield 3.45 g, 88%.
1
Mp 150–151ЊC, H NMR: 0.18 (dt, 2H), 0.46 (dt, 2H), 0.81
(t, 3H), 0.98 (m, 1H), 1.55 (sext, 2H), 2.42 (s, 3H), 3.28 (br t, 2H
ϩ 2H), 4.88 (s, 1H), 6.33 (br s, 1H NH), 7.41 (s, 2H); 13C NMR:
4.38, 10.42, 12.07, 14.53, 21.43, 50.80, 52.95, 79.14, 129.28,
132.96, 133.69, 135.09, 160.79, 162.55, 170.71. HRMS: (M ϩ
H)ϩ = 431.06122; calcd, 431.06315 (35Cl).
Mp 43–44 ЊC, 1H NMR: 2.6 (s, 3H), 7.08 (s, 1H); 13C NMR:
15.15, 116.47, 162.03, 175.17. GCMS: 195.1; calcd 195.1.
4-Chloro-2-methylmercapto-6-(2Ј,4Ј,6Ј-trichloroanilino)-
pyrimidine (11a). Synthesized using the general procedure as for
(10a). The brown solid was recrystallized from ethyl acetate–
hexanes to give a white solid, yield 360 mg, 60%.
2,4-Dichloro-6-methylpyrimidine (8). Synthesized using the
general procedure as for (6). Recrystallization with MeOH gave
white crystals, yield 4.18 g, 64%.
1H NMR: 2.48 (s, 3H), 5.90 (s, 1H), 7.21 (br s, 1H), 7.48 (s,
2H); 13C NMR: 14.78, 99.04, 129.51, 131.57, 134.73, 135.74,
160.90, 161.85, 173.31. HRMS: (M ϩ H)ϩ = 353.91987; calcd,
353.91937 (35Cl).
Mp 46–47 ЊC, 1H NMR: 2.57 (s, 3H), 7.20 (s, 1H); 13C NMR:
24.50, 112.29, 120.15, 163.01, 172.49. GCMS: 163.0; calcd
163.0.
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2-Chloro-6-methyl-4-(2Ј,4Ј,6Ј-trichloroanilino)pyrimidine
(16a) and 4-chloro-6-methyl-2-(2Ј,4Ј,6Ј-trichloroanilino)pyrim-
idine (14a). Synthesized using the general procedure as for
(10a). The yellow solid was recrystallized from ethyl acetate–
hexanes to give a pale yellow solid. Separation of the isomers
using flash chromatography with 1 : 4 ethyl acetate–hexanes
gave pale yellow solids, yield 14a 187 mg, 29%; 16a 363 mg,
56%.
acetate–hexanes. Yield 20c 100 mg, 53% as a yellow oil; 18c
30 mg, 27% as a white solid, mp 130–131 ЊC.
20c: 1H NMR: 0.21 (m, 2H), 0.44 (m, 2H), 0.82 (t, 3H), 1.06
(m, 1H), 1.56 (m, 2H), 2.19 (s, 3H), 3.38–3.42 (m, 4H), 5.48
(2, 1H), 5.99 (s, 1H), 7.40 (s, 2H); 13C NMR: 3.49, 10.14, 11.47,
20.93, 24.44, 49.18, 51.43, 92.10, 128.30, 132.05, 133.34, 134.58,
161.07, 166.98.
18c : 1H NMR: 0.1 (br s, 2H), 0.45 (br s, 2H), 0.75–1.02 (m,
4H), 1.50 (m, 2H), 2.26 (s, 3H), 3.14–3.26 (m, 4H), 5.82 (s, 1H),
6.60 (br s, 1H), 7.37 (s, 2H); 13C NMR: 3.52, 9.57, 11.29, 20.23,
24.29, 49.75, 51.79, 100, 127.61, 130.65, 134.85, 135.08, 162.16,
165.18.
14a 1H NMR: 2.38 (s, 3H), 5.92 (s, 1H), 7.53 (2H), 7.87 (br s,
1H); 13C NMR: 24.68, 101.57, 129.64, 131.69, 134.94, 136.00,
160.56, 163.35, 170.07. HRMS: (M ϩ H)ϩ = 186.07941; calcd,
186.07987 (35Cl).
16a 1H NMR: 2.29 (s, 3H), 6.63 (s, 1H), 7.25 (br s, 1H, NH),
7.39 (s, 2H); 13C NMR 24.42, 112.70, 129.12, 133.08, 133.35,
135.48, 160.61, 162.08, 170.69. HRMS: (M ϩ H)ϩ = 186.07946;
calcd, 186.07987 (35Cl).
2,4-Dichloro-5-methylpyrimidine (9). Synthesized using the
general procedure as for (6) with the exception that the extrac-
tion solvent used was dichloromethane. The product is was a
white solid or pale yellow oil (at RT), yield 4.69 g, 72%.
1
Mp 25ЊC; H NMR: 2.32 (s, 3H), 8.4 (s, 1H); 13C NMR:
2-Chloro-6-methyl-4-(N-propylamino)pyrimidine (16b) and
4-chloro-6-methyl-2-(N-propylamino)pyrimidine (14b). Syn-
thesized using the general procedure as for (10b). The yellow
solid was purified by flash chromatography using 15 : 85 ethyl
acetate–hexanes gave the products as yellow oils, yield 14b 23
mg, 50%; 16b 25 mg, 7%. Ratio of isomers 14b : 16b is 7.5 : 1.
16b 1H NMR: 0.85 (t, 3H), 1.56 (sex, 2H), 2.27 (s, 3H), 3.24
(br s, 2H, -CH2-NH-), 5.77 (br s, 1H), 6.06 (s, 1H); 13C NMR:
11.16, 22.23, 23.61, 43.19, 99.10, 159.92, 164.10, 167.53;
HRMS: (M ϩ H)ϩ = 202.09245; calcd, 202.09552 (37Cl).
16.32, 129.63, 158.71, 160.59, 163.05. GCMS: 163.0; calcd
163.0.
2-Chloro-5-methyl-4-(2Ј,4Ј,6Ј-trichloroanilino)pyrimidine
(17a). Synthesized using the general procedure as for (10a). The
product was obtained as an off-white gum. The product was
stirred with a hot 1 : 1 ethyl acetate–tetrahydrofuran solution
and was filtered giving a cream solid, with only one isomer
detected by TLC. The product was not purified by chromato-
graphy or recrystallization due to its low solubility in all
solvents. Yield 168 mg, 68%.
14b 1H NMR: 0.89 (t, 3H), 1.71 (sex, 2H), 2.27 (s, 3H), 3.07
(d, 2H), 5.63 (br s, 1H), 6.10 (s, 1H); 13C NMR: 11.63, 20.4,
1H NMR: 2.29 (s, 3H), 7.65 (s, 2H), 8.09 (s, 1H); 13C NMR:
13.09, 114.21, 125.77, 129.02, 133.83, 136.31, 157.06, 157.44,
162.10.
24.08, 53.69, 110.9, 158.0, 169.5, 170.8. HRMS: (M ϩ H)ϩ
=
200.09576; calcd, 200.09552 (35Cl).
2-Chloro-6-methyl-4-(N-isobutylamino)pyrimidine (16c) and
4-chloro-6-methyl-2-(N-isobutylamino)pyrimidine (14c). Syn-
thesized using the general procedure as for (10b). The product
was recrystallized from hexane and the isomers separated by
flash chromatography using 1 : 3 ethyl acetate–hexanes to give
off-white crystals, yield 14c 101 mg, 41%; 16c 57 mg, 23%. Ratio
of isomers 14c : 16c is 1.8 : 1.
2-Chloro-5-methyl-6-(N-propylamino)pyrimidine (17b). Syn-
thesized using the general procedure as for (10a). The second
isomer was not present by TLC after the reaction. The brown
solid was purified using a speedy column using 1 : 3 ethyl
acetate–hexanes to give a cream solid, yield 17b 105 mg, 46%.
1H NMR: 0.96 (t, 3H), 1.64 (m, 2H), 1.96 (s, 3H), 3.46 (dt,
2H), 4.68 (br s, 1H, NH), 7.76 (s, 1H); 13C NMR: 11.33, 12.94,
22.59, 42.86, 111.66, 154.51, 158.77, 162.33. HRMS: (M ϩ H)ϩ
= 186.07984; calcd, 186.07987 (35Cl).
14c 1H NMR: 0.88 (d, 6H), 1.81 (m, 1H), 2.23 (s, 3H), 3.18 (t,
2H), 5.32 (br s, 1H, NH), 6.35 (s, 1H); 13C NMR: 20.08, 23.77,
28.30, 48.82, 108.85, 160.97, 162.34, 169.37. HRMS: (M ϩ H)ϩ
= 240.12670; calcd, 240.12682 (35Cl).
2-Chloro-5-methyl-6-(N-isobutylamino)pyrimidine (17c) and
6-chloro-5-methyl-2-(N-isobutylamino)pyrimidine (15c). Syn-
thesized using the general procedure as for (9). The brown oil
was purified using a speedy column using 1 : 3 ethyl acetate–
hexanes to give the isomers as yellow oils, yield 17c 185 mg,
50%; 15c 25 mg, 6.8%. Ratio of isomers 17c : 15c is 7.4 : 1.
1
16c H NMR: 0.90 (d, 6H), 1.81 (m, 1H), 2.27 (s, 3H), 3.04
(br s, 2H), 5.32 (br s, 1H, NH), 6.03 (s, 1H); 13C NMR: 20.07,
23.79, 28.17, 49.14, 98.82, 160.25, 164.23, 167.89. HRMS: (M
ϩ H)ϩ = 240.12504; calcd, 240.12682 (35Cl).
1
2-Chloro-6-methyl-4-(N-propyl-N-cyclopropylmethylamino)-
pyrimidine (16d) and 4-chloro-6-methyl-2-(N-propyl-N-cyclo-
propylmethylamino)pyrimidine (14d). Synthesized using the
general procedure as for (10b). The brown oil was purified using
a speedy column using 1 : 3 ethyl acetate–hexanes to give the
isomers as yellow oils, yield 16d 135 mg, 46%; 14d 35 mg, 12%.
Ratio of isomers 16d : 14d is 3.8 : 1.
17c H NMR (CDCl3): 0.82 (d, 6H), 1.80 (m, 1H), 1.88 (s,
3H), 3.20 (t, 2H), 5.18 (br s, 1H, -NH-), 7.61 (s, 1H); 13C NMR:
13.93, 21.00, 28.95, 49.20, 112.92, 154.98, 159.30, 163.39.
HRMS: (M ϩ H)ϩ = 200.09578; calcd, 200.09552 (35Cl).
1
15c H NMR (CDCl3): 0.87 (d, 6H), 1.78 (m, 1H), 2.13 (s,
3H), 3.25 (t, 2H), 5.10 (br s, 1H, –NH–), 8.05 (s, 1H); 13C NMR:
13.95, 20.95, 29.08, 49.86, 104.88, 155.43, 157.54, 164.59.
GCMS: 200.1; calcd, 200.1.
16d 1H NMR: 0.28 (dd, 2H), 0.46 (dd, 2H), 0.90 (t, 3H), 1.06
(m, 1H), 1.62 (sext, 2H), 2.25 (s, 3H), 3.48 (d, 2H), 3.58 (t, 2H),
6.30 (s, 1H); 13C NMR: 3.55, 9.93, 11.37, 20.79, 23.97, 49.34,
51.81, 107.27, 160.68, 161.71, 168.71.
2-Chloro-5-methyl-6-(N-propyl-N-cyclopropylmethylamino)-
pyrimidine (17d) and 4-chloro-5-methyl-2-(N-propyl-N-cyclo-
propylmethylamino)pyrimidine (15d). Synthesized using the
general procedure as for (10b). The brown oil product was
purified by a speedy column using 10 : 10 : 80 ethyl acetate–
chloroform–hexanes, resulting in two yellow oils, yield 17d 185
mg, 50%. 15d 25 mg, 7%. Ratio of isomers 17d : 15d is 7.5 : 1.
17d 1H NMR: 0.12 (dt, 2H), 0.40 (dt, 2H), 0.78 (t, 3H), 0.90
(m, 1H), 1.50 (sext, 2H), 2.10 (s, 3H), 3.23 (d, 2H), 3.37 (t, 2H),
7.63 (s, 1H); 13C NMR: 4.11, 10.43, 11.51, 18.60, 22.01, 51.56,
54.49, 113.28, 157.97, 159.41, 164.08. GCMS: 239.1; calcd
239.1.
14d 1H NMR: 0.21 (dd, 2H), 0.46 (dd, 2H), 0.87 (t, 3H), 0.97
(m, 1H), 1.56 (sext, 2H), 2.29 (s, 3H), 3.34 (d, 2H), 3.40 (t, 2H),
6.10 (s, 1H); 13C NMR: 3.58, 9.36, 11.19, 20.29, 23.82, 49.91,
52.17, 99.37, 159.99, 162.80, 166.67.
6-Methyl-2-(N-propyl-N-cyclopropylmethylamino)-4-(2Ј,4Ј,6Ј-
trichloroanilino)pyrimidine (20c) and 6-methyl-4-(N-propyl-N-
cyclopropylmethylamino)-2-(2Ј,4Ј,6Ј-trichloroanilino)pyrimidine
(18c). Synthesized using the general procedure as for (12a). The
pale yellow solid was purified by flash column using 1 : 3 ethyl
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 3 5 3 – 3 3 6 1
3360

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15d 1H NMR: 0.25 (dt, 2H), 0.44 (dt, 2H), 0.88 (t, 3H), 1.03
(m, 1H), 1.59 (sext, 2H), 2.06 (s, 3H), 3.43 (d, 2H), 3.52 (t, 2H),
8.00 (s, 1H); 13C NMR: 4.21, 10.52, 12.08, 15.85, 21.36, 50.10,
52.57, 115.73, 159.37, 161.19, 161.40. GCMS: 239.1; calcd
239.1.
15 T. W. Lovenberg, D. E. Grigoriadis, D. T. Chalmers, J. R. McCarthy
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inhibitors, subtypes, pharmacology, localization, and their role in
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16 J. F. Hernandez, W. Kornreich, C. Rivier, A. Miranda,
G. Yamamoto, J. Andrews, Y. Tache, W. Vale and J. Rivier, Synthesis
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17 D. W. Schulz, R. S. Mansbach, J. Sprouse, J. P. Braselton, J. Collins,
M. Corman, A. Duaiskis, S. Faraci, A. W. Schmidt, T. Seeger,
P. Seymour, J. D. Tingley III, E. N. Winston and Y. L. Chen, Heym,
CP-154,526 - A potent and selective nonpeptide antagonist of
corticotropin releasing factor receptors, Proc. Natl. Acad. Sci. USA,
1996, 93, 10477–10482.
Acknowledgements
We are grateful for financial support from the NH&MRC
Australia. We also thank Dr Dimitri Grigoriadis for con-
ducting CRH binding assays.
18 A. McCluskey, M. Finn, M. Bowman, P. A. Keller and R. Smith,
3,5-Dimethyl-7-hydrazinothiazolo[4,5-d]pyradazine-7-thiol: A corti-
cotrophin releasing hormone type 1 receptor agonist, Aus. J. Chem.,
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19 J. P. Whitten, Y. F. Xie, P. E. Erickson, T. R. Webb, E. B. D. Souza,
D. E. Grigoriadis and J. R. McCarthy, Rapid microscale synthesis, a
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