Synthesis and antimycobacterial activity of 5-formylaminopyrimidines; analogs of antibacterial purines

Article abstract of DOI:10.1016/j.bmcl.2009.04.082

Pyrimidine analogs of antimycobacterial purines have been synthesized and their biological activities evaluated. Several 5-formamidopyrimidines exhibited profound activity against Mycobacterium tuberculosis in vitro (IC₉₀ ≤ 1.5 μg/mL), and they

Full text of DOI:10.1016/j.bmcl.2009.04.082

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3297–3299
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antimycobacterial activity of 5-formylaminopyrimidines;
analogs of antibacterial purines
Morten Brꢀ
ndvang a, Colin Charnock b, Lise-Lotte Gundersen a,
*
a Department of Chemistry, University of Oslo, PO Box 1033, Blindern, N-0315 Oslo, Norway
b Faculty of Health Sciences, Oslo University College, PO Box 4 St. Olavs plass, N-0130 Oslo, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrimidine analogs of antimycobacterial purines have been synthesized and their biological activities
Received 4 March 2009
Revised 14 April 2009
Accepted 17 April 2009
Available online 23 April 2009
evaluated. Several 5-formamidopyrimidines exhibited profound activity against Mycobacterium tubercu-
losis in vitro (IC90 6 1.5
l
g/mL), and they were essentially inactive against other bacteria.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Pyrimidine
Antimycobacterial
Tuberculosis
Ring-opening
Tuberculosis (TB) still claims ca. 2 mill. deaths pr year world-
wide and resistance to existing drugs is a growing problem.1 We
have previously studied 6-aryl-9-benzylpurines as antimycobacte-
rial agents.2 Structures of some purines with profound antimyco-
bacterial activity as well as a summary of SAR knowledge are
shown in Figure 1. These compounds display several properties
which make them highly interesting as potential drugs against
tuberculosis such as high selectivity towards Mycobacterium tuber-
culosis (Mtb) compared to other microorganisms, activity against
several drug resistant strains of Mtb, generally low toxicity towards
mammalian cells, and ability to affect Mtb inside macrophages.
After exploring SAR of intact purines,2 we decided also to study
non-purine analogs3 of the compounds described above in order
identify the real pharmacophore. We herein report synthesis and
antimycobacterial activity for pyrimidine analogs with the general
structure shown in Figure 1.
We have previously found that 2-nitropurines are valuable syn-
thetic intermediates,2f,4 and the 2-nitropurine 1a can be converted
to the 2-oxopurine 3 (Scheme 1).2f A careful study of this reaction
revealed that the ring-opening product 2a was formed initially,
and after 1 h the pyrimidine 2a could be isolated in 70% yield. After
a prolonged reaction time, compound 2a disappeared and the only
product present was compound 34 (Scheme 1). The oxopurine 3
may have been formed via an elusive unstable species 4, arbitrary
drawn as one possible tautomer. Alternatively, there is an equilib-
rium between purine 1a and 2a and a slow irreversible reaction of
compound 1a to oxopurine 3.
Since the formamidopyrimidine 2a was readily available by a
ring-opening of the corresponding purine 1a (Scheme 1), we sub-
jected a series of purines 1 to the same set of reaction conditions
(Table 1).5 Compounds 1 chosen as starting materials, generally
exhibit profound antimycobacterial activity,2 and the products 2
are thus 5-formamidopyrimidine analogs of antimycobacterial
purines. Compounds 1a and 1c–1e, carrying electron withdrawing
substituents in the purine 2-position, participated readily in the
ring-opening reaction and >90% conversion was generally seen
after 1 h. In case of the 2-nitropurine 1a and the 2-fluoropurine
1c, minor amounts of the 2-oxopurine 3 were also formed, due
to facile substitution in the 2-position. Without an activating
group at C-2, the reaction was much slower and for the purines
1b and 1f–1i an 18 h reaction time was required in order to get
the desired ring-opening products 2 in reasonable yields. The least
reactive purine was compound 1i carrying a powerful electron do-
nor (NMe2) in the 2-position. In the case of purine 1i, prolonged
reaction time (66 h) did not improve the yield of 2i.
Due to restricted rotation around the amide bond in the 5-form-
ylaminopyrimidines 2, two rotamers were generally observed in the
NMR spectra, with the s-cis rotamer as the major form in DMSO-d6.
NH-CHO coupling constants were in the area of 11–12 Hz for the s-
trans rotamer and ca. 1 Hz, when observed, for the s-cis rotamers.
These values are in good agreement with coupling constants found
for other 5-formylaminopyrimidines.6 At ambient temperature the
s-cis: s-trans ratios were ca. 8:2 with only minor variations in the ra-
tio depending on the pyrimidine 2-substituent.
* Corresponding author. Tel.: +47 22857019; fax: +47 22855507.
E-mail address: l.l.gundersen@kjemi.uio.no (L.-L. Gundersen).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.082

3298
M. Brꢀndvang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3297–3299
Figure 1. Structures of some potent antimycobacterial purines, summary of SAR knowledge, and general structure of pyrimidines described herein.
Scheme 1. Reagents: (a) Bu4NOH, H2O, THF.
Table 1
Synthesis of 5-formamidopyrimidines 2 by ring-opening of purines 1
R2
Time (h)
Yield 2a (%)
Recovered 1a (%)
NO2
H
F
Cl
CF3
CH3
CH2CH3
OCH3
N(CH3)2
1
18
1
1
1
18
18
18
18
70, 2a
66, 2b
72, 2c
81, 2d
59, 2e
44, 2f
32, 2g
65, 2h
14, 2i
0, 1ab
3, 1b
0, 1cc
5, 1d
7, 1e
26, 1f
53, 1g
12, 1h
85, 1i
Scheme 2. Reagents and conditions: (a) p-CH3O–C6H4–CH2NH2, Et3N, n-BuOH,
(b) (2-Furyl)SnBu3, (Ph3P)2PdCl2, DMF, 90 °C.
D;
a
Yield of isolated compound.
b
11% of the 2-oxopurine 6 was present in the crude product according to 1H
NMR.
c
7% of the 2-oxopurine 6 was present in the crude product according to 1H NMR.
are included in Figure 1. The values found for the pyrimidines,
are comparable to those found earlier for the corresponding pur-
ines, and both in the purine2f and pyrimidine series small and
lipophilic substituents (i.e., F, Cl, CH3) in the 2-position are beni-
ficial for the inhibition of Mtb growth.
Even though formamidopyrimidines 2a–2k are synthetically
available from the corresponding purines 1, one cannot exclude
that the ring-opening process may be reversed in the bioassay used
to determine antimycobacterial activity. Hence, any bioactivity ob-
served from formamidopyrimidines may actually (in part) be
caused by the parent purine 1. However, NMR studies indicate that
most formamidopyrimidines were not prone to cyclization. Espe-
cially pyrimidines with electron withdrawing substituents in the
2-position were highly stable and these compounds were also
the most active antimycobacterials.
In order to evaluate the importance of the formamido group for
biological activity, we also chose to synthesize pyrimidines 2j and
2k by reaction of the dichloropyrimidines 5a–b with p-methoxy-
benzylamine followed by (2-furyl)tributyltin under Stille condi-
tions (Scheme 2).
The furylpyrimidines 2 were screened for antibacterial activ-
ity against M. tuberculosis H37Rv in vitro and the results are pre-
sented in Table 2.7 Except for the dimethylaminopyrimidine 2i,
profound activities were found for all formamidopyrimidines,
whereas the pyrimidines 2j and 2k, lacking the formamide func-
tionality, were essentially inactive. IC90 values for the purines 1b
and 1d, corresponding to the formamidopyrimidines 2b and 2d,

M. Brꢀ
ndvang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3297–3299
3299
Table 2
Antibacterial and cytotoxic data for pyrimidines 2a
Compd
R2
R5
IC90 M. tuberculosis H37Rv (
l
g/mL)b
IC50 M. tuberculosis H37Rv (
l
g/mL)b
MIC S. aureus (
l
g/mL)c
MIC E. coli (
l
g/mL)d
2a
2b
2c
2d
2e
2f
2g
2h
2i
NO2
H
F
NHCHO
NHCHO
NHCHO
NHCHO
NHCHO
NHCHO
NHCHO
NHCHO
NHCHO
H
1.1
0.59
0.22
<0.20
<0.20
0.26
<0.20
0.26
0.53
11
>64
>64
>64
>64
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
>64
>64
>64
>64
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.56
<0.20
0.20
0.58
0.33
0.53
1.5
Cl
CF3
CH3
CH2CH3
OCH3
N(CH3)2
H
26
2j
2k
n.d.e
n.d.e
n.d.
n.d.
H
NH2
a
General structure of pyrimidines 2 is shown in Figure 1.
IC90 amicain 0.13 and IC50 amicain 0.07 lg/mL.
b
c
MIC gentamycin 0.1
lg/mL.
d
e
MIC gentamycin 0.5
lg/mL.
0% inhibition of Mtb at 6.25 lg/mL.
In accordance with previous findings on the structurally related
References and notes
purines,2 the pyrimidines exhibit a selective antimycobacterial
1. See for instance: Bhowruth, V.; Dover, L. G.; Besra, G. S. Prog. Med. Chem 2007, 45,
169. and references therein.
2. (a) Bakkestuen, A. K.; Gundersen, L.-L.; Langli, G.; Liu, F.; Nolsøe, J. M. J. Bioorg.
Med. Chem. Lett. 2000, 10, 1207; (b) Gundersen, L.-L.; Nissen-Meyer, J.; Spilsberg,
B. J. Med. Chem. 2002, 45, 1383; (c) Andresen, G.; Gundersen, L.-L.; Nissen-Meyer,
J.; Rise, F.; Spilsberg, B. Bioorg. Med. Chem. Lett. 2002, 12, 567; (d) Bakkestuen, A.
activity. Compounds 2a–2d were essentially inactive against
Staphylococcus aureus and Escherichia coli (MIC > 64
2).8 The nitropyrimidine 2a was also examined for cytotoxicity
against VERO cells,9 and the EC50 value was found to be >40
g/
mL (62% cell viabiliy at 40 g/mL).
lg/mL, Table
l
l
K.; Gundersen, L.-L.; Utenova, B. T. J. Med. Chem. 2005, 48, 2710; (e) Br
ꢀndvang,
Except for some similarities with antimycobacterial purines
previously described by us,2 the potent pyrimidines discussed
herein has no structural resemblance with any other compounds
studies as (potential) antimycobacterial drugs. This may indicate
that the pyrimidines do not act by the same mechanism as any
existing TB-drug, and such drug leads are preferred since there is
an increased possibility that resistance will not be developed
within a reasonable time frame. Furthermore, the pyrimidines
are easily synthesized and they comply with the Lipinski rule.
The latter is a good indication that they will display acceptable
oral availability.
M.; Gundersen, L.-L. Bioorg. Med. Chem. 2005, 13, 6360; (f) Br
Gundersen, L.-L. Bioorg. Med. Chem. 2007, 15, 7144.
ꢀndvang, M.;
3. Br
4. Br
ꢀ
ndvang, M.; Gundersen, L.-L. Tetrahedron Lett. 2007, 48, 3057.
ꢀndvang, M.; Gundersen, L.-L. Synthesis 2006, 2993.
5. General procedure for the synthesis of pyrimidines 2a–2i: Compound
1 was
dissolved, by heating if necessary, in THF (8 mL). The mixture was stirred at
ambient temperature and tetrabutylammonium hydroxide (0.67 mL, 1.0 mmol,
1.5 M sol. in water) was added over 2 min. The reaction mixture was stirred for
the time given in Table 1. In case of compounds 2b and 2f–2i, a small amount of
silica gel was added, the mixture evaporated and the product isolated by flash
chromatography on silica gel. In case of compounds 2a and 2c–2e, the reaction
mixture was poured into satd aq NH4Cl (30 mL). The resulting mixture was
extracted with EtOAc (2 Â 30 mL), the combined organic layers were washed
with water (2 Â 30 mL) and brine (30 mL), dried (MgSO4) and evaporated. The
products were purified by flash chromatography on silica gel.
6. (a) Raoul, S.; Bardet, M.; Cadet, J. Chem. Res. Toxicol. 1995, 8, 924; (b) Fenn, M. D.;
Lister, J. H. J. Chem. Soc., Perkin Trans. 2 1976, 29.
Acknowledgments
7. a The pyrimidines were screened for antimycobacterial activities essentially as
described in Ref. 1 Compounds were tested in ten twofold dilutions, from
Antimycobacterial data were provided by the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility (TAACF)
through a research and development contract with the US National
Institute of Allergy and Infectious Diseases. Financial support from
The Norwegian Research Council (KOSK II, grant number 177368)
is also gratefully acknowledged.
100 lg/mL to 0.19 lg/mL, against Mycobacterium tuberculosis H37 Rv (ATCC
27294) in BACTEC 12B medium using the Microplate Alamar Blue Assay (MABA).
The IC90 and IC50 values are determined from the dose–response curve as the
IC90 using the curve fitting program XLFIT, formula 205.; (b) Collins, L. A.;
Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.
8. The antimicrobial data (S. aureus and E. coli) were obtained as described before:
(a) Vik, A.; Hedner, E.; Charnock, C.; Samuelsen, Ø.; Larsson, R.; Gundersen, L.-L.;
Bohlin, L. J. Nat. Prod. 2006, 69, 381; (b) Vik, A.; Hedner, E.; Charnock, C.; Tangen,
L. W.; Samuelsen, Ø.; Larsson, R.; Bohlin, L.; Gundersen, L.-L. Bioorg. Med. Chem.
2007, 15, 4016.
Supplementary data
9. The pyrimidines were screened for mammalian cell cytotoxicity to VERO cells
essentially as described in Ref. 1: After 72 h exposure, viability is assessed using
the CellTiter 96Ò Non-Radioactive Cell Proliferation Assay (MTT) reagent from
Promega. Cytotoxicity is determined from the dose–response curve as the EC50
using the curve fitting program XLFIT, formula 205.
Supplementary data (analytical data for novel compounds)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmcl.2009.04.082.