Identification of aminopyrimidine regioisomers via line broadening effects in1H and13C NMR spectroscopy

Article abstract of DOI:10.1071/CH03316

Substituted mono- and diamino-pyrimidines were synthesized as part of our medicinal chemistry programmes. Primary amines substituted at the 4-position exhibited room-temperature line broadening effects in both ¹H and ¹³C NMR spectros

Full text of DOI:10.1071/CH03316

Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2004, 57, 1079–1083
Identification of Aminopyrimidine Regioisomers via Line Broadening
Effects in 1H and 13C NMR Spectroscopy
James Garner,A Tim Hill,A Luke Odell,A Paul Keller,B Jody Morgan,B
and Adam McCluskeyA,C
A Chemistry,School of Environmental and Life Sciences,University of Newcastle,Callaghan NSW 2308,Australia.
B Department of Chemistry, University of Wollongong, Wollongong NSW 2522, Australia.
C Corresponding author: Email: Adam.McCluskey@newcastle.edu.au
Substituted mono- and diamino-pyrimidines were synthesized as part of our medicinal chemistry programmes.
1
Primary amines substituted at the 4-position exhibited room-temperature line broadening effects in both H and
13C NMR spectroscopy due to the presence of rotamers, but these effects were not observed for substituents in
the 2-position. This provided a simple diagnostic tool for the identification of regioisomers, a determination which
would otherwise have required two-dimensional experiments.
Manuscript received: 12 December 2003.
Final version: 8 June 2004.
Introduction
Results and Discussion
Substituted pyrimidines are an important class of com-
pounds among medicinal chemistry programmes due to their
relative ease of manipulation and prevalence as effective
antagonists/agonists in biological systems.[1,2] These hetero-
cycles are amongst the most extensively investigated organic
molecules. We are currently engaged in varied medicinal
chemistry research programmes requiring simple mono- and
disubstituted aminopyrimidines. For example, we have inves-
tigatedtheirutilityascompetitiveantagonistsofcorticotropin
releasing hormone.[3,4]
Crucial to these synthetic studies was the development of
simple and rapid protocols for complete characterization of
the different regioisomers that might form during our syn-
thetic transformations. Surprisingly, there are few literature
precedents to facilitate rapid assignment of regiochemistry.
The majority of NMR investigations of simple pyrimidines
were performed during the early era of NMR structure elu-
cidation on low-resolution instruments, and were usually
without reported carbon spectra.[5–7] These investigations
focussed on compound sets that provided detailed data on
tautomeric pyrimidines. Dynamic NMR studies have in some
instances indicated varying degrees of spectral line broaden-
ing, an effect that is almost exclusively reported for large pro-
tein fragments and nucleoside-derived structures.[8,9] Herein
we report on a surprisingly simple and ubiquitous methodol-
ogy for the assignment of regiochemistry in a small library
of aminopyrimidines.
The majority of compounds examined in this study were syn-
thesized by a 5 M lithium perchlorate–diethyl ether (LPDE)
mediated displacement.[10–12] Briefly, dichloropyrimidines
A–D (Table 1) were stirred overnight at room temperature in
5 M LPDE with excess aliphatic amine (5–10 equiv.); extrac-
tive workup gave, in all instances, monosubstituted prod-
ucts in moderate-to-good yields (25–64%). Regioisomers
of asymmetrical pyrimidines (C and D) afforded products
with a higher selectivity of C4 substitution over C2, typically
in ratios of 7–10 : 1. Cyclic aliphatic amines did not react
in this system; however, these derivatives were synthesized
by nucleophilic substitution using NaH in tetrahydrofuran
(THF). Disubstituted products were synthesized by reflux-
ing in THF, and required separation from monosubstituted
∗
products, affording only moderate yields.[13]
C4-monosubstituted derivatives of A, B, and C exhib-
ited significant room-temperature line broadening in both
the 13C NMR (at C5) and 1H NMR spectra (at the
α-carbon of the amine substituent). No such effects were
observed, with the C2 regioisomer facilitating unambiguous
assignment of the relative regiochemistry (Scheme 1), thus
obviating HMBC and HMQC experiments.† Line broadening
was noted for various linear and cyclic aliphatic amines and
benzylamine. Derivatives of secondary amines, anilines, e.g.,
2,4,6-trichloroaniline, and the derivatives of compound D did
not exhibit any line broadening irrespective of substitution
pattern. Diaminopyrimidines did not exhibit line broadening
∗ We have previously reported the experimental details for compounds A–D, 1–3, 5–7, 20–25, 27–33, 39, and 40. The experimental details for all other
compounds may be found in the Accessory Materials.
† Assignment of C2 or C4 substituents were confirmed by HMBC and HMQC experiments, and in all instances confirmed the above assignments based on
line broadening effects.
© CSIRO 2004
10.1071/CH03316
0004-9425/04/111079

1080
J. Garner et al.
Table 1. Pyrimidines synthesized including their parent compounds and method of synthesis
Methods: (a) 5 M LPDE, room temp., 24 h; (b) neat, room temp., 24 h; (c) NaH/THF, reflux, 18 h; (d) EtOH, sealed tube,
160◦C, 18 h; (e) THF, sealed tube, 160◦C, 18 h; (f) H2O, reflux, 72 h; (g) THF, 50◦C, 24 h
N
R4
R3
R1
N
R2
Parent
R1
R2
R3
R4
Method
Broad
Parent compounds
A
B
C
D
—
—
—
—
CH3
SCH3
Cl
Cl
Cl
Cl
Cl
H
H
H
CH3
Cl
Cl
CH3
H
—
—
—
—
—
—
—
—
Cl
Derivatives of symmetrical pyrimidines
1
2
3
4
5
6
7
8
A
A
A
B
B
B
B
B
B
—
—
CH3
CH3
CH3
SCH3
SCH3
SCH3
SCH3
SCH3
SCH3
H
PrnNH
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
a
a
a
d
a
a
a
c
c
f
yes
yes
no
yes
yes
yes
no
yes
yes
yes
yes
BuiNH
PrnNCH2Prc
EtnNH
PrnNH
BuiNH
PrnNCH2Prc
c-hexylNH
c-pentylNH
c-hexylNH
c-hexylNH
9
10
11
Cl
Cl
H
PrnNCH2Prc
f
Derivatives of asymmetrical pyrimidines (diagnostic)
12
13
14
15
16
17
18
19
20
21
22
23
24
25
C
C
C
C
C
C
C
C
C
C
C
C
C
C
(Me)2NCH2CH2NH
HOCH2CH2NH
Cl
HOCH2CH2NH
c-hexylNH
Cl
(Me)2NCH2CH2NH
Cl
HOCH2CH2NH
HOCH2CH2NH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
e
g
g
e
f
yes
no
yes
yes
no
yes
no
yes
no
yes
no
yes
no
Cl
c-hexylNH
Cl
f
n-hexylNH
Cl
a
a
a
a
a
a
e
e
n-hexylNH
Cl
PrnNH
Cl
PrnNH
Cl
BuiNH
Cl
BuiNH
Cl
BnNH
Cl
BnNH
yes
Other compounds
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
C
C
C
D
D
D
D
D
C
C
C
C
C
C
C
N-morpholino
Cl
N-morpholino
PrnNCH2Prc
Cl
H
H
H
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
CH3
CH3
CH3
H
H
H
b
a
a
a
a
a
a
a
e
e
f
no
no
no
no
no
no
no
no
yes
yes
yes
no
no
no
no
PrnNCH2Prc
Cl
PrnNH
Cl
BuiNH
Cl
BuiNH
Cl
PrnNCH2Prc
Cl
H
H
PrnNCH2Prc
PrnNCH2Prc
(Et)2N
Cl
c-hexylNH
c-hexylNH
(2,3-Me2)c-hexylNH
(2,3-Me2)c-hexylNH
(2,3-Me2)c-hexylNH
2,4,6-TCA
Cl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
PrnNCH2Prc
(Et)2N
Cl
e
e
c
c
2,4,6-TCA
from a primary amine derivative if the other amine was an
aniline, but it was observed when the group was an alicyclic
or aliphatic amine.
While simple pyrimidines with tautomeric groups have
been studied extensively in the last 20 years, there is no
evidence that pyrimidines with simple amine substituents
will tautomerize except via protonation, a process usually
facilitated by the addition of trifluoroacetic acid or methane-
sulfonic acid.[2] We therefore hypothesized that the observed
line broadening was a result of the presence of rotamers.

Identification of Aminopyrimidine Regioisomers
1081
R3
N
R1
higher temperatures occurred over a narrower range than in
CDCl3. In our attempts to explore our hypothesis, detailed
conformational analysis using Spartan 02 on regioisomers
22 and 23 was conducted. We believed that this would aid in
explaining the line broadening observed.§
2
N
5
Substituents
on C2 induce
no line broadening
4
R2
Primary substituents
on C4 induce line
broadening at C5
As illustrated in Fig. 2, regioisomer 22, which did not
exhibit line broadening, has fewer distinct conformations
(Fig. 2, top) than 23, which displays significant line broad-
ening (Fig. 2, bottom). There was only a small difference
between the calculated maximum and minimum energy con-
formers (approx. 7 kJ mol−1) of 22. In each conformation
the amine N H bond was calculated to be in the plane of the
pyrimidine ring, while the rest of the alkyl chain exhibited
substantial variation between each conformation.
R
R
R
1 ϭ SCH3, CH3, or amine
2 ϭ Cl or amine
3 ϭ CH3, Cl, or amine
Scheme 1. Requirements for line broadening with amino-substituted
pyrimidines.
A previous structural and conformational study of large
substituted triazines with allylamine and piperazine func-
tional groups observed line broadening effects analogous to
that observed in the pyrimidines.[14,15] Restricted rotation of
the Ar N bond was observed at room temperature in the
1H, 13C, and 15N NMR spectra. In contrast to the pyrim-
idines, 13C NMR line broadening was only observed from
the α-carbon of the amine substituent. Line broadening was
also observed from the 15N NMR spectrum at N3 and N5.
These nitrogens in the triazine ring are spatially analogous
to C5 of the pyrimidine ring and the results were in par-
allel to our line broadening observations in the 13C NMR
spectrum. Coupled with crystal structures of the mono- and
bis-methanesulfonates, it was determined that the line broad-
ening was a result of the equilibrium between two (or more)
rotamer conformations.[14,15]
As with other rotameric studies, variation of temperature
(up or down) affected NMR peak shape. The most signifi-
cant changes were observed in the 13C NMR spectrum for
the C5 peak. Broad doublets indicating a high- and low-
energy conformation were often observed at temperatures
below 298 K (Fig. 1a). At 313 K almost all the compounds
examined had passed the coalescence temperature (Tc)‡ and
exhibited a sharp peak due to the averaged environment. Only
large amine substituents induced 13C NMR line broadening
of the α-carbon of the amine substituent, but resolution into
two peaks was never observed over the temperature range
available (8–11, 17, 35, 36). A small degree of line broad-
ening was occasionally observed from C4 and C6 of the
pyrimidine ring.
The energy difference of 20 kJ mol−1 between the highest
and lowest energy conformers of 23 represents a reasonable
barrier to rotation at the temperatures over which line broad-
ening is observed. While more conformers were calculated,
they could be classified into three sets with distinctive fea-
tures. The high-energy conformers (11–14) always oriented
the amine N–H towards CH(5) and in the same plane as the
aryl ring (Fig. 2b, bottom). The low-energy conformers (1–5)
have the amine N–H oriented away from CH(5) and the
α-carbon of the amine in close proximity (Fig. 2a, bot-
tom).All other conformations orient the N–H towards CH(5),
though slightly out of plane with the aryl ring, with large dif-
ferences in the orientation of the alkyl chain. The relative
populations of each of the conformations enabling a clearer
picture of conformational mobility could not be calculated
within the program.
Primary aniline derivatives [e.g., 2,4,6-trichloroaniline
(2,4,6-TCA) 39] do not exhibit line broadening at room tem-
perature. This is explained by observations of a preferred
stable orthogonal orientation of the aryl ring of the aniline to
the plane of the pyrimidine ring.[16] Conformer distribution
calculations of 39 (data not shown) resulted in a 1.8 kJ mol−1
difference in energy between the highest and lowest energy
conformer. In all conformations the aniline ring was orthog-
onal to the pyrimidine ring; this provided a strong argument
to the previously reported single preferred conformation.
Analysis of the conformations calculated by Spartan 02
revealed that the high- and low-energy conformations
were analogous to the triazine crystal structures previously
reported.[14,15] The key difference, we propose, is that it is
the interactions between CH(5) of the pyrimidine ring and
the N–H group and α-carbon of the amine substituent that
induces the line broadening observed. Spartan 02 was able
to calculate bond lengths and angles with a high degree of
accuracy at high levels of theory, especially for relatively
simple compounds such as our set of pyrimidines. Calcu-
lated hydrogen distances between the highest and lowest
There was also a strong contribution to the rotamer equi-
librium by a C2 or C6 methyl (or SMe) substitution in our
compound set. Simple H substitution at C2 (10, 11) resulted
in coalescence temperatures that were significantly lower
(<283 K) than all other derivatives. Changing the solvent to
(CD3)2SO (Fig. 1b), using 4 as a model compound, signifi-
cantly perturbed the system so that an increased Tc of C5 was
observed. Additionally, the two C5 peaks at low temperature
were well resolved, and conversion into one sharp peak at
‡ Coalescence temperature, for the purpose of qualitative analysis, is reported as the temperature at which coalescence is actually observed; that is, a very
low, broad baseline hump is observed. Alternatively, Tc is estimated by extrapolating between the two measurements at which it occurred.
§ Compounds were geometry-optimized using the Møller–Plesset function at MP2 level of theory. Conformer distributions were calculated using molecular
mechanics at the MMFF level of theory. Compounds were rotated by 30◦ increments for all available torsion points of the C(aryl)–N bond of the amine
substituent. The program allowed for a maximum of 20000 conformations and a maximum ꢀE of 100 kJ mol−1 difference. Conformations that had similar
energies and van der Waals overlap were treated as identical by the programme, which reduced the set of ‘unique’ conformers reported. Each conformer can
be regarded as representative of a number of conformations with the same energy and similar structural space.

1082
J. Garner et al.
(a)
(b)
313K
303K
313K
298K
283K
292K
160
100
60
40
20
100
100
Fig. 1. Spectra of 17. (a) Effect of temperature on line broadening for C5 (approx. 100 ppm), α-carbon of amine (approx.
50 ppm), and other pyrimidine ring carbon (approx. 160 ppm) signals in CDCl3. (b) Effect of temperature increase on C5
signal of 4 in CDCl3 (left) and (CD3)2SO (right).
Ϫ420
Ϫ421
Ϫ422
(a)
Ϫ423
Ϫ424
Ϫ425
Ϫ426
Ϫ427
Ϫ428
1
2
3
4
5
6
7
8
9
(b)
Conformer
Ϫ220
Ϫ225
Ϫ230
Ϫ235
Ϫ240
Ϫ245
(a)
(b)
1
2
3
4
5
6
7
8
9 10 11 12 13 14
Conformer
Fig. 2. Top image depicts a graph showing the energy distribution of the conformers of 22: (a) the lowest energy conformer;
(b) the highest energy conformer. The bottom image depicts a graph showing the energy distribution of the conformers of 23:
(a) the lowest energy conformer; (b) the highest energy conformer.
H3C
N
N
Cl
H3C
H
N
N
Cl
energy conformers predicted a change from 2.44 to 2.51 Å
between H5 and the closest hydrogens (Scheme 2). While the
variation in distance is small, the hydrogens approach signif-
icantly closer than those observed by Amm et al. (from 3.0 to
3.8 Å).[14,15] Thecloserproximityissuggestiveofwhysignif-
icant room-temperature line broadening is observed for such
small molecules, compared to the much larger compounds in
which restricted rotation could be expected.
2.78Å
N
H
N
H
H
2.35Å
2.51Å
2.44Å
CH
H
Scheme 2. Calculated distances from the highest and lowest energy
conformers of 23.

Identification of Aminopyrimidine Regioisomers
1083
Conclusions
[3] P. A. Keller, M. Bowman, K. H. Dang, J. Garner, S. P. Leach,
R. Smith, A. McCluskey, J. Med. Chem. 1999, 42, 2351.
doi:10.1021/JM9900117
[4] P. A. Keller, L. Elfick, J. Garner, J. Morgan, A. McCluskey,
Bioorg. Med. Chem. 2000, 8, 1213. doi:10.1016/S0968-0896(00)
00074-2
[5] T. J. Delia, D. Stark, S. K. Glenn, J. Heterocycl. Chem. 1995,
32, 1177.
[6] A. R. Katritzky, J. M. Lagowski, Prototropic Tautomerism of
Heteroaromatic Compounds: II. Six-Membered Rings 1963,
p. 339 (Academic Press: New York, NY).
[7] A. R. Katritzky, J. M. Lagowski, Prototropic Tautomerism of
Heteroaromatic Compounds: I. General Discussion and Methods
of Study 1963, p. 311 (Academic Press: New York, NY).
[8] R. Stolarski, C. E. Hagberg, D. Shugar, Eur. J. Biochem. 1984,
138, 187.
[9] L. Dudycz, R. Stolarski, R. Pless, D. Shugar, Z. Naturforsch.
Sect. C: Biosci. 1979, 34, 359.
[10] A. Kumar, J. Org. Chem. 1994, 59, 4612.
C4-substituted pyrimidines with small primary aliphatic
and alicyclic functional groups exhibit line broadening in
1H and 13C NMR spectra at room temperature, with signif-
icant broadening observed from C5 of the pyrimidine ring.
This effect is usually reported for bulky pyrimidines with
molecular weights greater than 1000, and it is surprising
that instances of room-temperature line broadening of low
molecular weight pyrimidines have been overlooked, consid-
ering their preponderance in the literature. We believe such
broadening is due to rotamers arising from the hindered rota-
tion occurring between the CH(5) of the pyrimidine ring
and amine-substituent hydrogens. These observations have
allowed us to develop a simple and effective diagnostic tool
to determine the substitution pattern of different regioisomers
isolated during synthesis.
[11] S. Sankararaman, J. E. Nesakumar, Eur. J. Org. Chem.
2000, 2003. doi:10.1002/1099-0690(200006)2000:11<2003::
AID-EJOC2003>3.0.CO;2-C
Accessory Materials
[12] J. Garner, A. McCluskey, Heterocycl. Commun. 1999, 5, 503.
[13] A. McCluskey, P. A. Keller, J. Morgan, J. Garner, Org. Biomol.
Chem. 2003, 1, 3353. doi:10.1039/B305458F
[14] M. Amm, N. Platzer, J. P. Bouchet, J. P. Volland, Magn.
Reson. Chem. 2001, 39, 77. doi:10.1002/1097-458X(200102)
39:2<77::AID-MRC801>3.3.CO;2-8
[15] M. Amm, N. Platzer, J. Guilhem, J. P. Bouchet, J. P. Volland,
Magn. Reson. Chem. 1998, 36, 587. doi:10.1002/(SICI)1097-
458X(199808)36:8<587::AID-OMR347>3.3.CO;2-2
[16] C. N. Hodge, P. E. Aldrich, Z. R. Wasserman, C. H. Fernandez,
G. A. Nemeth, A. Arvanitis, R. S. Cheeseman, R. J. Chorvat,
et al., J. Med. Chem. 1999, 42, 819. doi:10.1021/JM980223O
General experimental methods, details for the syntheses of
compounds 4, 8–19, 26, and 34–38, and a typical example of
the identification of regioisomers by two-dimensional NMR
are available from the author or, until November 2009, the
Australian Journal of Chemistry.
Acknowledgments
This work was supported by the National Health and Medical
Research Council. J.G. and L.O. also thank the University of
Newcastle for financial support.
References
[1] R. Marek, A. Lycka, Curr. Org. Chem. 2002, 6, 35.
[2] A. J. Boulton, Comprehensive Heterocyclic Chemistry: the Struc-
ture, Reactions, Synthesis and Uses of Heterocyclic Compounds
1984 (Pergamon: Oxford).