Identification of regioisomers in a series of N-substituted pyridin-4-yl imidazole derivatives by regiospecific synthesis, GC/MS, and 1H NMR

Article abstract of DOI:10.1021/jo026619w

The regiospecific synthesis of 2a (Scheme 3), a novel and potent pyridinyl imidazole inhibitor of p38 MAP (mitogen-activated protein) kinase, and the regioselective preparation of its regioisomer 2b (Scheme 4) are described. Chromatographic and spectroscopic data are presented, which in this class of compounds allow the unambiguous identification of regioisomers prepared by a nonregiospecific synthetic strategy. Biological data demonstrating the importance of the correct regiochemistry for inhibition of p38 are given.

Full text of DOI:10.1021/jo026619w

similar heterocyclic) scaffold are required for efficient
binding to the target enzyme.8,9
Id en tifica tion of Regioisom er s in a Ser ies
of N-Su bstitu ted P yr id in -4-yl Im id a zole
Der iva tives by Regiosp ecific Syn th esis,
Because of the different substituents at the 4- and
5-positions of the imidazole nucleus placement of an
additional substituent at either one of the two imidazole
ring nitrogens generates two different regioisomers.
However, only at the ring nitrogen adjacent to the
pyridin-4-yl moiety (like in SB 210313, Figure 1) are
substituents tolerated without loss of p38 inhibition.2
With beneficial properties such as enhanced oral activ-
ity10 and reduced toxicity11 being ascribed to substituents
at the imidazole ring nitrogen, the regiospecific prepara-
tion of N1-substituted pyridin-4-yl imidazoles has been
addressed.1,12 However, if such N-substituted imidazoles
are prepared via a nonregiospecific synthetic pathway,
both regioisomers are usually formed3,13 and the assign-
ment of the correct regiochemistry becomes a challenging
task. In the structurally related class of 2,3-dihydroimi-
dazo[2,1-b]thiazole inhibitors of cytokine release, for
example, the molecular structures published for the
desfluoro congeners of regioisomers SK&F 86002 and
SK&F 86055 (Scheme 1) had been assigned on the basis
of spectroscopic data.14 The initial assignment, however,
was later disproved when X-ray crystallographic evidence
became available for these compounds.15 Like SK&F
86002 and SK&F 86055, methylsulfanylimidazoles ML
3375 and 1 (Figure 1) can be synthesized from suitable
imidazole-2-thione precursors (Scheme 1). Following the
discovery of 1H-imidazole 1 as a potent inhibitor of p38
MAP kinase and cytokine release,16 we sought to prepare
its N1-methylated analogue 2a (Scheme 3). Herein we
report the regiospecific synthesis of 2a as well as a fast
and convenient method to analytically distinguish the
N-substituted regioisomers of 2-methylsulfanylimidazole
derivatives using routine GC/MS and 1H NMR tech-
niques. This analytical protocol enabled us to unambigu-
ously determine the regioselectivity for the direct con-
1
GC/MS, a n d H NMR
Gerd K. Wagner,† Dunja Kotschenreuther,‡
Werner Zimmermann, and Stefan A. Laufer*
Institute of Pharmacy, Department of Pharmaceutical and
Medicinal Chemistry, Eberhard-Karls-University Tu¨bingen,
Auf der Morgenstelle 8, 72076 Tu¨bingen, Germany
stefan.laufer@uni-tuebingen.de
Received October 28, 2002
Abstr a ct: The regiospecific synthesis of 2a (Scheme 3), a
novel and potent pyridinyl imidazole inhibitor of p38 MAP
(mitogen-activated protein) kinase, and the regioselective
preparation of its regioisomer 2b (Scheme 4) are described.
Chromatographic and spectroscopic data are presented,
which in this class of compounds allow the unambiguous
identification of regioisomers prepared by a nonregiospecific
synthetic strategy. Biological data demonstrating the im-
portance of the correct regiochemistry for inhibition of p38
are given.
Several small molecule inhibitors of cytokine release
are currently being investigated for their potential as safe
and efficient antiinflammatory drugs. Work carried out
by various groups1-3 as well as in our own laboratory4,5
has identified polysubstituted imidazoles (Figure 1) as
potent inhibitors of p38 MAP kinase (for a recent review,
cf. J ackson and Bullington6). p38 is implicated in signal
transduction events leading to the release of proinflam-
matory cytokines interleukin 1â (IL-1â) and tumor
necrosis factor-R (TNF-R) from human monocytes,7 hence
the strong antiinflammatory effect exerted by p38 inhibi-
tors. Structure-activity relationships in this class of p38
inhibitors have revealed that the vicinal 4-fluorophenyl
and pyridin-4-yl moieties attached to an imidazole (or
* Author to whom correspondence should be addressed. Phone:
++49-7071-2972459. Fax: ++49-7071-295037.
(7) Lee, J . C.; Laydon, J . T.; McDonnell, P. C.; Gallagher, T. F.;
Kumar, S.; Green, D.; McNulty, D.; Blumenthal, M. J .; Heys, J . R.;
Landvatter, S. W.; Strickler, J . E.; McLaughlin, M. M.; Siemens, I. R.;
Fisher, S. M.; Livi, G. P.; White, J . R.; Adams, J . L.; Young, P. R. Nature
1994, 372, 739-746.
† Current address: Department of Pharmacy and Pharmacology,
University of Bath, Claverton Down, Bath BA2 4QN, United Kingdom.
‡ Current address: Ratiopharm GmbH, Department of Patent
Affairs, Graf-Arco-Strasse 3, 89079 Ulm, Germany.
(1) Boehm, J . C.; Smietana, J . M.; Sorenson, M. E.; Garigipati, R.
S.; Gallagher, T. F.; Sheldrake, P. L.; Bradbeer, J .; Badger, A. M.;
Laydon, J . T.; Lee, J . C.; Hillegass, L. M.; Griswold, D. E.; Breton, J .
J .; Chabot-Fletcher, M. C.; Adams, J . L. J . Med. Chem. 1996, 39, 3929-
3937.
(2) Gallagher, T. F.; Seibel, G. L.; Kassis, S.; Laydon, J . T.;
Blumenthal, M. J .; Lee, J . C.; Lee, D.; Boehm, J . C.; Fier-Thompson,
S. M.; Abt, J . W.; Soreson, M. E.; Smietana, J . M.; Hall, R. F.;
Garigipati, R. S.; Bender, P. E.; Erhard, K. F.; Krog, A. J .; Hofmann,
G. A.; Sheldrake, P. L.; McDonnell, P. C.; Kumar, S.; Young, P. R.;
Adams, J . L. Bioorg. Med. Chem. 1997, 5, 49-64.
(3) Liverton, N. J .; Butcher, J . W.; Claiborne, C. F.; Claremon, D.
A.; Libby, B. E.; Nguyen, K. T.; Pitzenberger, S. M.; Selnick, H. G.;
Smith, G. R.; Tebben, A.; Vacca, J . P.; Varga, S. L.; Agarwal, L.;
Dancheck, K.; Forsyth, A. J .; Fletcher, D. S.; Frantz, B.; Hanlon, W.
A.; Harper, C. F.; Hofsess, S. J .; Kostura, M.; Lin, J .; Luell, S.; O’Neill,
E. A.; Orevillo, C. J .; Pang, M.; Parsons, J .; Rolando, A.; Sahly, Y.;
Visco, D. M.; O’Keefe, S. J . J . Med. Chem. 1999, 42, 2180-2190.
(4) Laufer, S.; Striegel, H.-G.; Neher, K. PCT Int. Appl. 2000
(Merckle GmbH, Germany), WO 0017192, 2002, p 53.
(8) Tong, L.; Pav, S.; White, D. M.; Rogers, S.; Crane, K. M.; Cywin,
C. L.; Brown, M. L.; Pargellis, C. A. Nat. Struct. Biol. 1997, 4, 311-
316.
(9) Wang, Z.; Canagarajah, B. J .; Boehm, J . C.; Kassisa, S.; Cobb,
M. H.; Young, P. R.; Abdel-Meguid, S.; Adams, J . L.; Goldsmith, E. J .
Structure 1998, 6, 1117-1128.
(10) Adams, J . L.; Boehm, J . C.; Gallagher, T. F.; Kassis, S.; Webb,
E. F.; Hall, R.; Sorenson, M.; Garigipati, R. S.; Griswold, D. E.; Lee, J .
C. Bioorg. Med. Chem. Lett. 2001, 11, 2867-2870.
(11) Adams, J . L.; Boehm, J . C.; Kassis, S.; Gorycki, P. D.; Webb,
E. F.; Hall, R.; Sorenson, M.; Lee, J . C.; Ayrton, A.; Griswold, D. E.;
Gallagher, T. F. Bioorg. Med. Chem. Lett. 1998, 8, 3111-3116.
(12) Laufer, S.; Wagner, G.; Kotschenreuther, D. Angew. Chem., Int.
Ed. 2002, 41, 2290-2293.
(13) Chang, L. L.; Sidler, K. L.; Cascieri, M. A.; de Laszlo, S.; Koch,
G.; Li, B.; MacCoss, M.; Mantlo, N.; O’Keefe, S. J .; Pang, M.; Rolando,
A.; Hagmann, W. K. Bioorg. Med. Chem. Lett. 2001, 11, 2549-2553.
(14) Klose, W.; Schwarz, K. J . Heterocycl. Chem. 1985, 22, 669-
671.
(15) Shilcrat, S. C.; Hill, D. T.; Bender, P. E.; Griswold, D. E.;
Bauers, P. W.; Eggleston, D. S.; Lantos, I.; Pridgen, L. N. J . Heterocycl.
Chem. 1991, 28, 1181-1187.
(16) Laufer, S.; Wagner, G.; Kotschenreuther, D.; Albrecht, W. J .
Med. Chem. Submitted for publication.
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4695-4705.
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1011-1020.
10.1021/jo026619w CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/25/2003
J . Org. Chem. 2003, 68, 4527-4530
4527

F IGURE 1. Inhibitors of p38 MAP kinase and cytokine release. The essential pharmacophore for inhibition of p38 is highlighted.
SCHEME 1. Syn th esis of SK&F 86002 a n d SK&F
8605517
isolated as the main product. We finally managed to
obtain nitrile 3 from the condensation of the activated
isonicotinic acid analogue with 4-fluorophenylacetonitrile
in an aprotic solvent. Compound 3 was converted into
R-hydroximinoketone 5 via ketone 4 by standard proce-
dures. However, the acid-catalyzed hydrolysis and sub-
sequent decarboxylation of nitrile 3 was accompanied by
the cleavage of the amide bond and therefore necessitated
the reintroduction of the acetyl protecting group in a
separate step prior to cyclization.
Target compound 2a was obtained from R-hydroximi-
noketone 5 in a five-step synthesis (Scheme 3). Upon
treatment of 5 with 1,3,5-trimethylhexahydro-1,3,5-tri-
azine, the imidazole-N-oxide 6 was formed, which then
was converted in situ into imidazole-2-thione 7 utilizing
2,2,4,4-tetramethylcyclobutane-1,3-dithione19 as the sul-
fur source. S-Methylation of imidazole-2-thione 7 with
excess iodomethane afforded methylsulfanylimidazole 8,
and subsequent acid-catalyzed cleavage of the amide
bond in 8 furnished aminopyridine 9. The nucleophilic
displacement reaction of 1-phenylethylbromide with 9
provided target pyridinyl imidazole 2a .
SCHEME 2. P r ep a r a tion of Key In ter m ed ia te 5a
Although the requirement for an unambiguously es-
tablished regiochemistry at the imidazole core was fully
met by this reaction sequence, it suffered from several
drawbacks. Most importantly, yields were low to moder-
ate for the synthetic steps leading up to R-hydroximi-
noketone 5 and the protecting group approach was
required, as the free amino group was not tolerated
during the ring closure reaction. To facilitate the prepa-
ration of 2a as well as the rapid synthesis of various
analogues modified at the 2-position of the pyridine ring,
the direct conversion of 1 and its analogue 10 into 2a
and 11a was attempted (Scheme 4).
a
Reagents: (a) carbonyldiimidazole, absolute DMF, rt; (b)
potassium tert-butoxide, 4-fluoro-phenylacetonitrile, absolute DMF,
120 °C; (c) 48% hydrobromic acid, reflux; (d) acetic anhydride,
4-(dimethylamino)pyridine, reflux; (e) isoamyl nitrite, sodium
methoxide, methanol, rt.
Upon treatment of 116 with excess methyliodide in
DMF, N-methylation was expected to take place at either
imidazole ring nitrogen. Therefore, a reliable and fast
analytical method was needed to determine the ratio by
which regioisomers 2a and 2b were formed during the
course of this reaction. In a series of N1-alkylated
analogues of ML 3375, it was found that other than the
parent ML 3375 (mp 263 °C), these compounds are
comparatively low-melting (mp 100-170 °C, cf. Support-
ing Information for examples).20 They were evaporated
without decomposition at 250 °C and could thus be
analyzed by GC/MS.20 When monitoring the N-methyla-
tion of 1 by GC/MS, four peaks corresponding to two pairs
of mass spectra, with identical molecular ions of m/z 418
and 432, respectively, were observed. We reasoned that
not only one of the imidazole ring nitrogens but also the
phenylethylamino group had reacted with the methylat-
ing agent under these conditions and that both possible
version of several 1H-imidazoles into their N-substituted
analogues.
Previously, we have established a regiospecific syn-
thetic route toward N1-substituted pyridin-4-yl imidazole
derivatives, starting from suitable R-hydroximinoketone
precursors.12 As a prerequisite to extending the scope of
this strategy to the preparation of analogues bearing an
acylamino or alkylamino side chain at the pyridine ring,
acetylamino pyridine 5 was required (Scheme 2). The
synthesis we had devised for 5 in turn relied on the
availability of nitrile 3. Attempts to prepare 3 from the
corresponding ester of isonicotinic acid and 4-fluorophen-
ylacetonitrile according to the standard protocol for this
type of reaction (sodium alkoxide catalysis in alcoholic
solvent)18 failed. Under these conditions, 4-fluorophenyl-
acetonitrile appeared to undergo polymerization, while
the isonicotinic ester was saponified and the free acid was
(17) Lantos, I.; Bender, P. E.; Razgaitis, K. A.; Sutton, B. M.;
DiMartino, M. J .; Griswold, D. E.; Walz, D. T. J . Med. Chem. 1984,
27, 72-75.
(18) Lantos, I.; Gombatz, K.; McGuire, M.; Pridgen, L.; Remich, J .;
Shilcrat, S. J . Org. Chem. 1988, 53, 4223-4227.
(19) Elam, E. U.; Davis, H. E. J . Org. Chem. 1967, 32, 1562-1565.
(20) Kotschenreuther, D. Ph.D. Thesis, Eberhard-Karls-Universita¨t
Tu¨bingen, Tu¨bingen, Germany, 2002.
4528 J . Org. Chem., Vol. 68, No. 11, 2003

SCHEME 3. Regiosp ecific P r ep a r a tion of Regioisom er 2a a
a
Reagents: (a) 1,3,5-trimethylhexahydro-1,3,5-triazine, ethanol, reflux; (b) 2,2,4,4-tetramethylcyclobutane-1,3-dithione, DCM, rt; (c)
iodomethane, K2CO3, methanol, rt; (d) 10% HCl, reflux; (e) 1-phenylethylbromide, DMF, NaH, reflux.
SCHEME 4. Syn th etic Ap p r oa ch es tow a r d Regioisom er s 2a a n d 2ba
a
Reagents and conditions: (a) iodomethane, Cs2CO3, DMF, rt; (b) DMF-DMA, toluene, reflux (the yield is given for the conversion of
10 into 11b); (c) 1-phenylethylamine, reflux (the yield is given for the conversion of 11b into 2b).
TABLE 1. Selected 1H-13C Con n ectivities Obser ved in th e HMBC Sp ectr a of Regioisom er s 2a a n d 2b
2a
SCH3
NCH3
C3-H pyridine
C5-H pyridine
C-C-C-H
imidazole C2 (δ 144.2 ppm)a
imidazole C5 (δ 128.3 ppm)a
C-S-C-H
C-N-C-H
C-N-C-H
C-C-C-H
2b
SCH3
NCH3
C3/C5-H 4-F-ph
imidazole C2 (δ 146.4 ppm)a
imidazole C4 (δ 134.6 ppm)a
C-S-C-H
C-N-C-H
C-N-C-H
C-C-C-H
a
Cf. Table 2 for the numbering of the imidazole nucleus.
imidazole regioisomers had been formed for both the
secondary and the tertiary amine. However, the crude
reaction mixture contained less than 20% of the desired
1-phenylethylamino pyridine 2a , which eluted after 12.61
min, but contained 32% of the regioisomer 2b (retention
time 14.55 min). The former peak was unambiguously
assigned to 2a by comparison with the analytical data
obtained for an authentic sample of 2a from the regio-
specific pathway.
Simultaneous alkylation at the imidazole ring and the
phenylethylamino nitrogen was avoided by methylation
of 2-fluoropyridine 1016 with excess dimethylformamide
dimethylacetal (DMF-DMA) in toluene and subsequent
introduction of the side chain by SNAr (Scheme 4). Under
these conditions, N-methylation proceeded with remark-
able regioselectivity and isomer 11b was the only reaction
product that could be isolated, though a small amount
of 11a was detected by GC/MS (retention time 12.3 min,
Table 2). Subsequent nucleophilic substitution of inter-
mediate 11b with 1-phenylethylamine afforded pyridinyl
imidazole derivative 2b. The biological results for 2a and
2b confirmed that placement of an additional substituent
at the imidazole ring nitrogen adjacent to the 4-fluoro-
phenyl moiety is detrimental for p38 inhibition. Com-
pound 2a exceeded its regioisomer in potency by 2 orders
of magnitude (Scheme 4).
The regioselectivity of the methylation step providing
11b was elucidated by comparison of the analytical data
for the final product of this reaction sequence (i.e., 2b)
and for an authentic sample of 2a . On the nonpolar
J . Org. Chem, Vol. 68, No. 11, 2003 4529

TABLE 2. Selectivity Ra tios for th e Alk yla tion of Va r iou s N-Un su bstitu ted Im id a zoles
product code
R
conditions
11
-F
11
-F
11
-F
11
-F
MeI, NaH,
THF, rt
0:100c
12
13
-OHa
-OHa
DMF-DMA,
reflux
2:98b
MeI, Cs2CO3,
DMF, rt
7:93b
MeI, Na2CO3,
DMF, rt
7:93b
MeI, MeOH,
reflux
BzBr, Na2CO3,
EtOH, reflux
0:100c
ratio of a :b
0:100c
a
b
IR and 13C NMR data indicate that the pyridone is the preferred tautomeric form. Ratio determined by GC/MS. c Isomers 11b-13b
were isolated as the only reaction products in the following yields: 11b (36%), 12b (8%), 13b (3%).
stationary phase applied in GC/MS analysis, regioisomers
2a and 2b eluted over a time window of almost 2 min.
The observed elution order corresponded to the order of
melting points for 2a and 2b, as was expected, since
separation on nonpolar capillary phases is mainly at-
tributed to differences in vapor pressure of the solute.
For the unequivocal assignment of peaks, it was crucially
important to have a chromatographic system that clearly
distinguished between both regioisomers, not the least
because the mass spectra of 2a and 2b showed a very
similar pattern of fragmentation. For both regioisomers,
the base peak can be attributed to the molecular ion (m/z
418). The m/z 403 ion may result from demethylation at
either the methylsulfanyl or methylamino group. Cleav-
age of the phenylethylamino substituent in various
positions may account for the m/z 299, 120, and 105
fragments. Minor differences between the mass spectra
of both regioisomers concerned the intensity of peaks, the
most notable example being the relatively low abundance
of the m/z 120 ion in the mass spectrum of 2b.
The above assignment of the regioisomeric structures
of 2a and 2b is supported by the results from two-
dimensional NMR experiments. The NOESY spectrum
of 2a shows three cross-peaks for the N-methyl group (cf.
Supporting Information). Two of them result from the
interaction between the N-methyl protons and the C3-
and C5-protons of the pyridine ring. As expected from
the assigned structure, none of these cross-peaks is
present in the NOESY spectrum of regioisomer 2b. Here,
a single cross-peak for the N-methyl substituent indicates
its close proximity to the 4-fluorophenyl ring. The dif-
ferent connectivities observed in the respective HMBC
spectra of 2a and 2b are also in agreement with the
regiosomeric nature of these compounds (Table 1).
With a convenient GC/MS method at hand to monitor
the regioselectivity of the direct alkylation approach and
with the elution order of regioisomers 11a and 11b firmly
established, we employed a variety of reaction conditions
in order to improve the regioselectivity leading to the
desired isomer 11a (Table 2). However, a change in
neither the methylating agent (DMF-DMA vs MeI) nor
the strength of the base (NaH vs Cs2CO3) or the size of
the counterion (Na2CO3 vs CsCO3) considerably affected
regioselectivity. When the influence of the substituent
at the pyridine ring was tested in a series of pyridone
analogues, elevated reaction temperatures suppressed
N-substitution at the imidazole altogether. Here, 12b and
13b were the only isomers isolated in very poor yield,
regardless of the alkylating agent and the solvent (Table
2). In summary, the direct N-alkylation of the substituted
2-methylsulfanylimidazoles presented herein appears to
take place predominantly at the imidazole ring nitrogen
adjacent to the 4-fluorophenyl substituent. This is in
marked contrast to observations by other groups for
related C2 carba-substituted imidazole derivatives.3 How-
ever, the analytical protocol described above should
facilitate ongoing efforts to alter the regioselectivity of
this reaction.
With regard to the 1H NMR spectra of 2a and 2b,
regioisomeric substitution of the imidazole nucleus pre-
dominantly affected the chemical shifts for protons at the
substituted pyridine ring (cf. Supporting Information).
While an upfield shift by 0.59 and 0.32 ppm was observed
for the C3- and C5-protons, respectively, in 2a compared
to 2b, the signal assigned to the C6-proton in 2a was
shifted downfield by 0.61 ppm. As a result, the peaks
attributed to the pyridine ring protons spread over a
broad range of 2.06 ppm in the case of 2a compared to
only 0.87 ppm in the spectrum of 2b. The significantly
different distribution of electron density at the pyridine
ring of regioisomers 2a and 2b can be explained by the
different canonical forms induced by the presence of an
additional substituent at either one of the two imidazole
ring nitrogens. Therefore, we believe that in this class of
compounds, the range covered by the signals assigned
to the pyridine protons in the 1H NMR spectra is a
characteristic feature from which the regiochemistry of
the imidazole ring can be determined. This view is
supported by findings in a series of regioisomeric pyridi-
nyl imidazoles and pyridinyl 2,3-dihydroimidazo[2,1-b]-
thiazoles (cf. Supporting Information for examples).20 It
has to be noted that the differences in their 1H NMR
spectra are of particular value for the identification of
Ack n ow led gm en t. We thank K. Albert and P.
Schuler for providing the 600 MHz NMR data, Merckle
GmbH, Blaubeuren, Germany, and the Fonds der Che-
mischen Industrie for financial support, and C. Greim
for establishing the p38 test assay.
Su p p or tin g In for m a tion Ava ila ble: Detailed synthetic
procedures, spectroscopic data, and relevant examples from
reference 20. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
regioisomers, as only one isomer is required for H NMR
analysis, whereas both isomers are needed for compari-
son of GC retention times.
J O026619W
4530 J . Org. Chem., Vol. 68, No. 11, 2003