Novel pyridopyrazine and pyrimidothiazine derivatives as FtsZ inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.09.062

A series of pyridopyrazine and pyrimidothiazine derivatives have been synthesized and their activity against FtsZ from Mycobacterium tuberculosis (Mtb) and in vitro antibacterial activity against Mtb H₃₇Ra and Mtb H₃₇Rv are reported.

Full text of DOI:10.1016/j.bmc.2011.09.062

Bioorganic & Medicinal Chemistry 19 (2011) 7120–7128
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel pyridopyrazine and pyrimidothiazine derivatives as FtsZ inhibitors
Bini Mathew a, Shefali Srivastava a, Larry J. Ross a, William J. Suling a, E. Lucile White a, Lisa K. Woolhiser b,
Anne J. Lenaerts b, Robert C. Reynolds a,
⇑
a Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA
b Department of Microbiology, Immunology and Pathology, Colorado State University, 200 West Lake Street, CO 80523, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyridopyrazine and pyrimidothiazine derivatives have been synthesized and their activity
Received 22 September 2011
Accepted 29 September 2011
Available online 5 October 2011
against FtsZ from Mycobacterium tuberculosis (Mtb) and in vitro antibacterial activity against Mtb
H
37Ra and Mtb H37Rv are reported. Certain analogs described herein showed moderate to good inhibitory
activity.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Pyridopyrazine
Pyrimidothiazine
FtsZ inhibitor
Antitubercular
1. Introduction
N
N
HN
N
HN
Tuberculosis remains
a
high priority public health threat
N
N
Ph
Ph
N
N
Ph
throughout the world, particularly in developing nations. The
increasing prevalence of drug resistant strains of Mycobacterium
tuberculosis from single (SDR) to multiple (MDR), extensively
(XDR) and even totally (TDR) drug resistant strains underscores
concerns within the public health network of the developed world
and emphasizes the critical need for heightened disease surveil-
lance and increased basic research, particularly into pathogen biol-
ogy and new drug targets as well as greater emphasis on new drug
discovery with the goal of developing more effective combination
treatments.1,2
O
O
N
N
H
O
O
N
H
N
Ph
1
2
Figure 1. Structures of lead FtsZ inhibitors 1 and 2.
activity relationship (SAR) for the series against FtsZ with the even-
tual goal of improving potency against the target, increasing whole
cell antibacterial activity, and reducing off target toxicity.
Over the past decade, FtsZ (Filament temperature sensitive pro-
tein Z), the prokaryotic analog of tubulin, has received considerable
attention as a new antibacterial drug target. This protein plays an
essential role in bacterial cell division, and interruption of this
process is a bactericidal event. Several inhibitors have been re-
ported.3–10 Among these, an anti-malarial precursor 2-alkoxycar-
bonylaminopyridine, 1 (Fig. 1), was found to be a potent inhibitor
of Mtb growth as well as selectively inhibiting in vitro polymeriza-
tion of FtsZ relative to tubulin.11–13 A close pyrimidine analog 2
(Fig. 1) of the 2-alkoxycarbonylaminopyridines has also been re-
ported as an inhibitor of FtsZ polymerization, equipotent to 1 but
less effective against Mtb growth in vitro.12
2. Chemistry
Lead compounds 1 and 2 were prepared as reported and were
analytically identical with the original authentic samples.12,13 Syn-
thetically, 6–10 and 17–25 are accessible through one common
key intermediate 3.14 The synthetic route for 6–10 and 17–25 is
outlined in Scheme 1.
The pyridopyrazine analogs 6–10 were prepared by the dis-
placement of the 4-chloro group of 3 with 2-amino-5-diethylamin-
opentane to form 4.13 Reduction of the 5-nitro group of 4 yielded 5,
and direct coupling with different diketones gave the final targets
6–10 in good yields. Similarly for the synthesis of targets 17–25, the
displacement of the 4-chloro group of 3 with different amino com-
pounds afforded 11–13, and reduction of the 5-nitro group using
Raney nickel and H2 at atmospheric pressure and room tempera-
ture gave 14–16 in quantitative yields. Final coupling with benzil,
2,20-thenil or furil provided the targets 17–25 in good yields.
Herein, we report the synthesis of several pyridopyrazine and
pyrimidothiazine analogs in order to further develop a structure–
⇑
Corresponding author. Tel.: +1 205 581 2454; fax: +1 205 581 2877.
E-mail address: reynolds@southernresearch.org (R.C. Reynolds).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.062

B. Mathew et al. / Bioorg. Med. Chem. 19 (2011) 7120–7128
7121
N
Cl
R
HN
NO2
NH2
NO2
NH2
NO2
NH2
(a)
O
(a)
O
O
N
H
N
O
O
N
H
N
N
H
N
O
3
11-13
4
11: R= -HN(CH2)4N(Et)2
12
: R= -HN(CH2)2N(Et)2
(b)
(b)
13: R= -HN(CH2)2N(Me)2
N
R
HN
NH2
NH2
NH2
NH2
O
O
O
N
H
N
O
N
H
N
5
14-16
14: R= -HN(CH2)4N(Et)2
: R= -HN(CH2)2N(Et)2
: R= -HN(CH2)2N(Me)2
15
16
(c)
(c)
N
R
HN
N
N
R'
R'
N
R
R
O
O
O
N
H
N
O
N
N
N
H
6-10
17-25
17: R= -HN(CH2)4N(Et)2; R'= Phenyl
18
: R= -HN(CH2)4N(Et)2; R'= Thiophen-2-yl
6
7
: R= Furan-2-yl : R= Thiophen-2-yl
19: R= -HN(CH2)4N(Et)2; R'= Furan-2-yl
20: R= -HN(CH2)2N(Et)2; R'= Phenyl
8: R= Pyridin-2-yl 9: R= Methyl
10: R= Ethyl
21
: R= -HN(CH2)2N(Et)2; R'= Thiophen-2-yl
22: R= -HN(CH2)2N(Et)2; R'= Furan-2-yl
23: R= -HN(CH2)2N(Me)2; R'= Phenyl
24
: R= -HN(CH2)2N(Me)2; R'=Thiophen-2-yl
25: R= -HN(CH2)2N(Me)2; R'= Furan-2-yl
Scheme 1. Synthetic pathways to analogs 6–10 and 17–25. Reagents and conditions: (a) 2-amino-5-diethylaminopentane/4-diethylaminobutylamine/2-diethylaminoeth-
ylamine/2-dimethylaminomethylamine, MeOH/EtOH, reflux; (b) Raney Nickel, H2, EtOH, rt; (c) Benzil/Furil/2,20-Thenil/2,20-Pyridil/2,3-butanedione/3,4-hexanedione, EtOH,
reflux, N2.
R'
N
R'
N
3. Results and discussion
3.1. In vitro cell studies
N
N
R
R
N
N
R
R
O
(a)
O
N
H
H2N
26
26, 27
: R= Furan-2-yl
27: R= Thiophen-2-yl
6, 7
The minimum inhibitory concentration (MIC, the lowest con-
centration that completely inhibits growth) of all compounds for
Mtb H37Ra were determined using a colorimetric (Alamar blue)
microdilution broth assay reported previously.11 The compounds
were also screened against Mtb H37Rv to determine the IC90 (the
concentration that inhibits 90% of growth) as described previously
and Vero cell cytotoxicity (CC50).18 All the compounds were also
examined for their ability to inhibit the target, Mtb FtsZ, and its
mammalian homolog tubulin (reported as IC50 values) as described
previously.11 Screening data are given in Table 1.
R'= -HNCH(CH3)(CH2)3N(Et)2
Scheme 2. Synthetic pathway to 26–27. Reagents and conditions: (a) KOH, EtOH,
N2, reflux.
The carbamate groups in 6 and 7 were hydrolyzed by treatment
with KOH in ethanol under reflux to give 26 and 27, respectively in
moderate yields (Scheme 2).14
The synthetic route for 37 and 38 is outlined in Scheme 3.
Commercially available 2-amino-4,6-dichloropyrimidine was re-
fluxed with oxalyl chloride in benzene followed by addition of
ethyl alcohol to provide 4,6-dichloro-2-pyrimidyl urethane
(28).12,15 Compound 28 was refluxed with either 2-amino-5-
diethylaminopentane to yield 29 or 4-diethylaminobutylamine
to give 30, in quantitative yields. Compounds 29 and 30 were ni-
trated with sulfuric acid (conc.) and nitric acid (fum.) at 35 °C to
provide the 5-nitropyrimidines (31 and 32).12,16 Displacement of
the 4-chloro group in 31 and 32 by potassium thioacetate under
reflux gave 33 and 34.17 Reduction to the 5-aminopyrimidines 35
and 36 by refluxing with zinc in acetic acid at 80 °C followed by
condensation with desyl chloride in the presence of sodium ace-
tate under nitrogen atmosphere gave the target thiazines 37 and
38 respectively.17
SAR substitutions were driven by screening results of available
pyridopyrazines from the Southern Research repository versus Mtb
H
37Rv and comparison to published anticancer and tubulin screen-
ing results.11,19 Through our initial comparison, it was clear that
four positions altered antitubercular and FtsZ selectivity versus
Vero cells, mammalian cancer cells, and tubulin – see Figure 2.
Structure 39 represents the typical pyridopyrazine that would
inhibit both mammalian cancer cells and tubulin as described by
Temple.19 Certain features are important, if not crucial to antican-
cer and antimitotic activity, including a carbamate at R1, smaller
linear alkyl functions at R2 (acyl and larger heteroalkyl are not
tolerated), aryl functions (typically phenyl) at R3, and smaller alkyl
0
functions at R4 and/or R4 . Significant anticancer activity resides
with the fused pyrazine systems (X3 = N; O or S not tolerated),
although increasing (diazepine) or decreasing (imidazole) the size

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B. Mathew et al. / Bioorg. Med. Chem. 19 (2011) 7120–7128
R
R
N
Cl
NO2
Cl
(a)
N
O
N
O
N
(b)
O
N
H
N
O
N
H
O
N
H
N
Cl
O
Cl
29, 30
31, 32
28
R
N
R
N
R
NO2
SH
N
S
Ph
Ph
NH2
SH
(c)
(d)
O
N
N
O
O
(e)
N
H
N
O
N
H
O
N
H
N
O
33, 34
35, 36
37, 38
N
N
29, 31, 33, 35, 37 R = HN
30, 32, 34, 36, 38
HN
R =
Scheme 3. Synthetic pathways to pyrimido[4,5-b][1,4]thiazines 37–38. Reagents and conditions: (a) 2-amino-5-diethylaminopentane/4-diethylaminobutylamine, MeOH,
reflux; (b) fuming HNO3, concentrated H2SO4, 30–35 °C; (c) potassium thioacetate, EtOH, reflux; (d) Zn, AcOH, N2, 80 °C; (e) desyl chloride, NaOAc, H2O/EtOH, rt.
Table 1
Screening data for control 1 and 2, pyridopyrazine analogs 6–10, 17–25 and 26–27, and pyrimidothiazines 37–38a
Compounds Mtb FtsZ Polymerizationb IC50
(l
M) Tubulin Polymerizationb IC50
>100
24% inhibition 100
(l
M) Mtb H37Ra MIC (
l
M) Mtb H37Rv IC90
(l
M) Vero Cytotoxicity CC50 (lM)
1
2
6
7
34.2 2.5
38.1 4.1c
26.8 5.6
34.3 6.0
45.7 3.6
lM
0.47
3.8
3.6
0.43
54
>290
>290
0.24
0.23
3.6
1.7
67.9
8.7
<0.19
1.9
7.0
ND
ND
>40
>40
>40
>40
>40
1.8
>40
>40
2.7
6.3
5.5
0.7
1.6
37
l
M
>100
>100
>100
>100
>100
lM
lM
lM
lM
lM
<0.19
>100
>100
>50
<0.19
>100
>100
0.38
0.92
>100
<0.19
<0.19
29
8
9
>100
>100
52
l
l
M
M
10
17
18
19
20
21
22
23
24
25
26
27
37
38
40% inhibition 100
l
M
64% at 100
16.4 1.2
59% at 100
21.0 2.0
34.8 10.7
54.6 18.9
27.3 3.2
34.1 14.6
18.6 2.3
29.5 14.5
46.0 3.4
46.2 4.3
l
M
M
>100
lM
41 5.9
l
>100
>100
>100
>100
>100
>100
>100
>100
>100
lM
lM
lM
lM
lM
lM
lM
lM
lM
67.6
1.1
67.0
150
17
>100
4.1
22
14
5.9
>40
>40
49
52
69.7 3.4
18
IC50 = concentration to inhibit polymerization of either tubulin or FtsZ by 50%. MIC = minimum inhibitory concentration is the lowest concentration, using duplicate two-fold
serial dilutions of drug, that completely inhibited growth as evidenced by a lack of metabolic dye reduction in the microplate Alamar blue assay—see Ref. 11 IC90 is
extrapolated from a 10 point dose response curve and is the concentration that inhibits bacterial growth by 90%—see Ref. 18 CC50 (Cytotoxic Concentration) is extrapolated
18
from a 10 point dose response curve and is the concentration that inhibits growth of Vero cells in culture by 50%—see Ref.
.
a
Methods are reported in Refs. 11 and 18.
b
Each compound was analyzed at least three times per assay. For the polymerization assays, the mean the standard deviations are reported.
c
Reported previously.12
substituents in the carbamate group (R1), sterically demanding R2
NHR2
NHR2
groups (diphenylmethyl), as well as aromatization of the pyrido-
pyrazine and inclusion of larger aryl functions at R4 (see structures
1 and 2 and general structure 40 and discussion in Ref. 11). Thus,
we chose to examine certain relatively facile substitutions that
would scan the SAR pattern in these regions of structure 40 in or-
der to optimize whole cell activity and FtsZ/tubulin selectivity.
Hence, we initially explored the SAR of compound 1 at the 6,7
positions with heterocycles such as furan, thiophene and pyridine,
and small alkyl groups such as methyl and ethyl (6–10). These ana-
logs were designed to evaluate the role of hydrophobic and steric
differences in antitubercular activity. Among these five com-
pounds, compound 6 with two furan-2-yl rings at the 6,7-positions
of 1 was found to be modestly more FtsZ active than 1. The MIC of 6
5N
R3
4
3
N
R3
X2
6 R4'
X2
2
7
8
R1HN
1
X1 X3 R4
R1HN
X1
X3 R4
39
40
Figure 2. General structures of heterocyclic inhibitor scaffolds
of this fused pyrazine ring can also impact anticancer and tubulin
activity with the best inhibitors in the pyridodiazepine systems.20
Certain of these systems show significant antitubercular activity
and select examples also can impact FtsZ polymerization in vitro
(Ref. 11 and unpublished results). Beyond these overlaps in activ-
ity, there are clear alterations that significantly reduce tubulin
and anticancer activity in the series including larger (alkyl or aryl)
(3.6
(0.47
strain H37Rv than the lead sample 1. The thiophene-2-yl analog
l
M) for H37Ra was eight-fold greater than the MIC of 1
lM), although 6 appeared less active versus the virulent

B. Mathew et al. / Bioorg. Med. Chem. 19 (2011) 7120–7128
7123
7, in contrast, had activity very similar to 1 in all the screening as-
says. The pyridine analog 8 was less active than 1 against H37Ra
reduced lipophilicity and reduced passive transport through the
bacterial cell wall.
and H37Rv but showed good activity for FtsZ (ID50 of 45.7
l
M),
Finally, a modest set of two analogs were prepared to study the
effect of substituting a thiazine ring for the pyrazine ring in 2.
These analogs were targeted to determine if insertion of sulfur
for nitrogen would enhance target binding through an increase in
the S–C bond lengths and alteration of planarity of the aromatic
ring; such effects have been observed in tubulin binding with the
deazapteridines.19 Furthermore, certain pyridodiazepines showed
significant tubulin and cancer activity, and it was hypothesized
by Temple that the larger non-planar diazepine ring allowed better
interaction of the 6-Ph group with contact points in the tubulin
structure. These analogs also showed significant antitubercular
activity and inhibition of FtsZ polymerization.11 Hence, we decided
to prepare the S (thiazine) for N (pyrazine) substitution to deter-
mine the effects of interrupting the aromaticity of the pyrazine
ring by adding the larger S atom that cannot participate equiva-
suggesting that the more polar analog may have cell wall perme-
ability issues. The less hydrophobic compounds 9 and 10, with
methyl and ethyl groups at the 6,7-positions respectively, were
inactive against FtsZ and both bacterial strains in vitro.
As previously discussed, the C-4 position is a critical area for
modification in terms of activity and selectivity. While a diphenyl-
methyl substitution at the 4-position of the bicyclic system
showed significant and selective antitubercular and FtsZ activity
in earlier screens, there were concerns regarding the medicinal
properties of such hydrophobic and high molecular weight ana-
logs. Hence, we chose to focus efforts on the 4-diamino alkyl
substituted series that also showed relatively high activity and
selectivity in our screens. Initially, efforts centered on the active
6,7-disubstituted phenyl, furan and thiophene scaffolds, and we
explored variations in chain length [–N–(CH2)2–N vs –N–(CH2)4–
N] and removal of the branched methyl group to ascertain the ef-
fects of this stereocenter on activity. Additionally, the terminal
dialkylamino group was substituted with either a dimethyl or a
diethyl substituent in these analogs. Compounds 17–25, with
N,N-diethylaminobutylamino group (17–19—these analogs lack
chirality and the Me group), N,N-diethylaminoethylamino group
(20–22—these analogs are shorter chain species and lack chirality)
and N,N-dimethylaminoethylamino group (23–25—shorter chain
analogs lacking chirality and substituting a dimethylamino for a
diethylamino group in the lead compounds) at the 4-position,
had activity against H37Ra and H37Rv similar to that of 1, 6 and 7
with the exception of 18 and 19, which were inactive against
lently with the trivalent N atom in the pyrazine p-system; it would
be expected that this substitution might allow the proposed puck-
ering of the thiazine ring resulting in the 6-Ph being out of plane
and altering both the tubulin and possibly the FtsZ interaction.
Both samples contain chiral centers; 37 has two stereocenters
while 38 contains one chiral center at position 7. The MIC of 37
and 38 for Mtb H37Ra were 49 and 52
to the MIC (H37Ra) reported previously for 2 (3.8
37 and 38 were 16-fold higher. Compounds 37 and 38 displayed
IC90 values of 21.7 and 18.5 M against H37Rv, 10-fold less active
l
M, respectively. Compared
l
M),12 the MIC of
l
than the lead compound 2. In contrast, 37 and 38 were only
slightly less potent than 2 in their inhibition of FtsZ polymerization
(ID50 of 46 lM for both 37 and 38 compared to 38 lM for 2). It is
H
37Rv. Compounds 1 and 6 compare to analogs 17 and 19, respec-
notable that 38 lacks the Me group that generates a chiral center
in the C-2 side chain, and that sample showed significant tubulin
activity as was noted with other similar samples (17 and 19) that
lacked the Me appendage at this position again suggesting that
the Me group at this point may play some role in selectivity for FtsZ
inhibition. Ideally, further pursuit of many of these structures,
including both 37 and 38, would require separation of complex
mixtures of stereoisomers in order to obtain conclusive SAR com-
parisons, and the relatively active shorter chain (and achiral) ana-
logs at C-2 are attractive in that regard. A mammalian cell
cytotoxicity screen was also run in order to assess relative selectiv-
ity of the reported compounds for bacterial inhibition. While there
are interesting data points suggesting that FtsZ can be inhibited
while not impacting tubulin polymerization, for the most part
compounds that show antibacterial activity are also cytotoxic to
Vero cells; this result lends further credence to the hypothesis that
there are additional, off target activities of the class.
tively, which are basically the same compounds minus the
branched methyl group that produces a stereocenter in com-
pounds 1 and 6. In both cases it is notable that the two samples
(17 and 19) are relatively comparable in all screens relative to
the parent samples (1 and 6), although 19 is relatively less active
versus avirulent Mtb and inactive versus Mtb H37Rv. More interest-
ing is the fact that both 17 and 19 show modest activity in the
tubulin polymerization assay, possibly suggesting that the methyl
group may play a role in selectivity against FtsZ. It is also interest-
ing that the comparable disubstituted thiophen-2-yl analog 18 is
significantly less potent than the methyl containing parent 7 in
all screens as well as the tubulin polymerization assay. Compounds
20–22, containing the shorter C-2 side chains all retained FtsZ
polymerization activity, although only 21 and 22 were equipotent
with the parent 6,7-difuran-2-yl and 6,7-dithiophen-2-yl analogs,
suggesting that shorter chain analogs can, depending on other sub-
stitutions on the heterobicycle, retain target activity. It is notable
that 20–22 all retain significant antitubecular activity, although
22 was virtually inactive against Mtb H37Rv. Compounds 23–25
contained the C-2 –N–(CH2)2–NMe2 and all samples showed rela-
tively similar activity to 20–22 with this modest structural alter-
ation. It is notable that 20–25 with the shortened C-2 side chain
In summary, we report herein further SAR analysis of the pyri-
dopyrazine lead series that shows significant antitubercular activ-
ity and inhibition of the novel target FtsZ.
3.2. In vivo animal studies
did not show any tubulin polymerization activity up to 100
l
M
Concurrently with the SAR studies, both compounds 1 and 7
were selected for further evaluation in vivo in order to determine
cytotoxicity and efficacy in a murine Mtb model to evaluate these
scaffolds as further candidates for antitubercular drug discovery
against FtsZ. The toxicity of compounds was studied in an acute
maximum tolerated dose C57BL/6 mouse model (MTD) to evaluate
toxicity and determine dose for safe use in mouse studies. Mice
were administered escalation doses up to 300 mg/kg for 3 days
daily by gavage with the compounds formulated in 0.5% methyl
cellulose, and dosages were scaled back if any mortality or adverse
effects were observed. Once an acceptable MTD value was deter-
mined, the compounds were tested for in vivo efficacy in a short
term mouse M. tuberculosis model. This model uses the Inter-
regardless of the fact that they did not contain the stereocenter
of the lead compounds, giving synthetic options in designing inhib-
itors that do not have chirality/separation issues.
We also prepared a small set of 2-NH2 analogs (26–27) in order
to probe the requirement for a carbamate at the 2-position. These
samples are comparable to the 6,7-difuran-2-yl and 6,7-dithio-
phen-2-yl analogs, 6 and 7 respectively. Compounds 26 and 27,
formed by the hydrolysis of 6 and 7, were relatively comparable
to 6 and 7 for FtsZ polymerization inhibition with IC50 values of
18.6 and 29.5 lM respectively. Neither of these samples showed
tubulin polymerization activity, and both showed reduced activity
in the whole bacterial growth assays, possibly a result of modestly

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B. Mathew et al. / Bioorg. Med. Chem. 19 (2011) 7120–7128
feron-
c
gene-disrupted C57BL/6 mice (GKO) and was developed
gene, these
consumption of the starting material 3, the reaction mixture was
and extensively tested.21 Without the protective IFN-
c
cooled to room temperature and evaporated to dryness under re-
duced pressure. Diethyl ether was added drop wise to the residue
and it was refrigerated for crystallization. The resulting solid was
filtered and washed with ether (2 ꢀ 1 mL) and chilled ethanol
(2 ꢀ 1 mL) to give 11 (110 mg, 47%) as a yellow solid. mp 178–
180 °C. TLC (10% MeOH–CHCl3–1% NH4OH): Rf 0.38. 1H NMR
(300 MHz, CDCl3): d 9.18–9.10 (m, 1H, NH), 7.02 (s, 1H, NH), 6.76
(s, 1H, 5-H), 4.22 (q, 2H, J = 7.2 Hz, OCH2), 3.38 (dd, 2H, J = 6.9 Hz,
12.6 Hz, NH–CH2), 3.17–2.97 (m, 6H, 3 ꢀ NCH2), 2.00–1.95 (m,
2H, CH2), 1.84–1.78 (m, 2H, CH2), 1.40 (t, 6H, J = 6.3 Hz, 2 ꢀ CH3),
1.31 (t, 3H, J = 6.9 Hz, CH3). MS (ES) m/z (M+H)+ 369.
mice are highly susceptible to the M. tuberculosis infection and,
therefore, the activity of a compound can be seen rapidly when
compared to untreated controls. This sensitive mouse model re-
quires only 9 days of treatment and a small number of animals,
which makes it an ideal model for first line testing of TB com-
pounds. The compounds were formulated in 0.5% methyl cellulose
and administered daily by gavage. The viable counts were con-
verted to logarithms, which were then evaluated by a one-way
analysis of variance, followed by a multiple comparison analysis
of variance by a one-way Tukey test (SigmaStat software program).
Differences were considered significant at the 95% level of confi-
dence. The lead compound of the pteridine series, compound 1,
was initially evaluated for in vivo toxicity using an acute toxicity
mouse model. This compound showed lethality at 300 mg/kg and
significant adverse effects at 100 and 30 mg/kg. The dosage se-
lected for further in vivo efficacy studies of compounds in this ser-
ies was therefore selected to be 10 mg/kg. In the short term GKO
mouse model, 1 showed significant efficacy in the lungs by reduc-
ing the bacterial load by 0.86Log10 CFU (P <0.05) whereas the
compound did not show any significant activity in the spleen
(0.5log10 CFU reduction, P >0.05). For compound 7, there was no
in vivo activity observed in lungs as well as spleens in the GKO
mouse model after 9 days of treatment at 10 mg/kg (reduction of
0.51Log10 CFU in lungs, and 0.58Log10 CFU in spleens) (P >0.05).
In conclusion, compound 1 showed efficacy at a very low dose
of 10 mg/kg, which shows that the series might show some prom-
ise if the toxicity issue could be resolved. We have, however, deem-
phasized this class of compounds due to concern with the
significant toxicity of the pyridopyrazine leads in the animal mod-
els. This fact, coupled with the relative lack of activity against tubu-
lin suggests that there may be off target activities of the class that
have yet to be determined. Furthermore, the relative difficulty of
preparation, the high molecular weights of the active products
and the poor dynamic range in the FtsZ SAR have led us to pursue
other FtsZ inhibitors discovered through our screening programs,
and these will be reported in due course.
4.2. Ethyl-6-amino-4-[[2-(diethylamino)-ethyl]-amino]-5-nitro-
2-pyridinecarbamate (12)
The procedure was followed as detailed above using 3 (173 mg,
0.66 mmol) and N,N-diethylethylenediamine (0.38 mL, 2.65 mmol).
The product was purified by silica gel column chromatography
(0.5% MeOH–CHCl3–1% NH4OH) to afford 12 (200 mg, 88%) as a yel-
low solid. TLC (10% MeOH–CHCl3–1% NH4OH): Rf 0.63. 1H NMR
(300 MHz, CDCl3): d 9.45 (bs, 1H, NH), 6.91 (s, 1H, NH), 6.74 (s,
1H, 3-H), 4.26 (q, 2H, J = 6.9 Hz, OCH2), 3.33–3.28 (m, 2H, NH–CH2),
2.75 (t, 2H, J = 6.3 Hz, NH–CH2–CH2), 2.60 (q, 4H, J = 6.9 Hz, -NCH2),
1.34 (t, 3H, J = 6.9 Hz, –OCH2–CH3), 1.07 (t, 6H, J = 7.2 Hz, –NCH2–
CH3). MS (ES) m/z (M+H)+ 341.
4.3. Ethyl-6-amino-4-[[4-(dimethylamino)-ethyl]-amino]-5-
nitro-2-pyridinecarbamate (13)
The procedure was followed as detailed above for 11 using 3
(1.5 g,
5.7 mmol),
N,N-dimethylethylenediamine
(0.76 mL,
6.9 mmol) and methanol (20 mL). The title compound 13 (1.27 g,
71%) was obtained by silica gel column chromatography (0.5%
MeOH–CHCl3–1% NH4OH) as a yellow solid. mp 238–240 °C. TLC
(10% MeOH–CHCl3–1% NH4OH): Rf 0.63. 1H NMR (300 MHz,
DMSO-d6): d 9.97 (s,1H, NH), 9.05 (t, 1H, J = 5.4 Hz, NH), 8.00 (bs,
2H, NH2), 6.69 (s, 1H, 3-H), 4.17 (q, 2H, J = 6.9 Hz, OCH2), 3.65–
3.62 (m, 2H, NH–CH2), 3.29–3.26 (m, 2H, NH–CH2–CH2), 2.79 (bs,
6H, –NCH3), 1.25 (t, 3H, J = 7.2 Hz, –OCH2–CH3). MS (ES) m/z
(M+H)+ 313.
4. Experimental
Anhydrous solvents and reagents from Aldrich were used
without further drying. Reactions were monitored by thin-layer
chromatography (TLC) on precoated E. Merck silica gel (60F254)
plates (0.25 mm) and visualized using UV light (254 nm). Flash
chromatography was carried out on Fischer silica gel G 60
(230–400 mesh). Melting points, determined with a Mel-Temp II
capillary melting points apparatus, are uncorrected. 1H NMR
spectra were recorded on a Nicolet NT 300NB instrument at
300 MHz. The coupling constants (J) are reported in hertz, and
chemical shifts are reported in ppm (d) relative to residual sol-
vent peak or internal standard. ESI-MS spectra were recorded
on a BioTof-2 time-of-flight mass spectrometer. Mass spectra
(HRMS) were recorded on an Agilent 6210 LCMS-TOF instrument.
The purity of the synthesized compounds was determined on a
Agilent 1100 hplc instrument using gradient system on a vydac
column.
4.4. General procedure for the synthesis of 5, 14–16
A solution of carbamate in ethanol was hydrogenated at room
temperature and 1 atm pressure in the presence of Ra-Ni (washed
3 ꢀ H2O and 3 ꢀ EtOH) for 7–8 h. Progress of the reaction was
monitored by TLC. On completion of the reaction, the catalyst solu-
tion was filtered through celite and the filtrate was evaporated to
dryness yielding a quantitative amount of diamine. These com-
pounds were directly used in the next step without further
purification.
4.5. Ethyl (8-((5-(diethylamino)pentan-2-yl)amino)-2,3-
di(furan-2-yl)pyrido[2,3-b]pyrazin-6-yl)carbamate (6)
Furil (74 mg, 0.39 mmol) was added to a stirred solution of the
diamine hydrochloride 5 (140 mg, 0.35 mmol) in ethanol (10 mL).
The clear yellow solution was stirred at room temperature under
nitrogen atmosphere for 24 h, and then refluxed for 16 h under
the same conditions. The solvent was removed in vacuo leaving a
residual solid, which was purified with silica gel column chroma-
tography using 6% MeOH–CHCl3–1% NH4OH as eluant. The frac-
tions containing compound were concentrated under vacuum to
give 6 (120 mg, 66%, HPLC Purity: 96.3%) as a yellow solid. mp
65–70 °C. TLC (10% MeOH–CHCl3–1% NH4OH): Rf 0.51. 1H NMR
4.1. Ethyl-6-amino-4-[[4-(diethylamino)-butyl]-amino]-5–
nitro-2-pyridinecarbamate hydrochloride (11)
4-Diethylaminobutylamine (91 mg, 0.63 mmol) was added to a
stirred solution of 3 (150 mg, 0.57 mmol) in dry ethanol (5 mL) and
the resulting solution was refluxed under nitrogen atmosphere.
Progress of the reaction was monitored by TLC. After the complete

B. Mathew et al. / Bioorg. Med. Chem. 19 (2011) 7120–7128
7125
(300 MHz, CDCl3): d 7.58 (dd, 1H, J = 0.9 Hz, 1.8 Hz, 50-H), 7.53 (dd,
2H, J = 0.9 Hz, 1.8 Hz, 50-H, NH), 7.45 (s, 1H, 6-H), 6.87 (dd, 1H,
J = 0.9 Hz, 3.3 Hz, 30-H), 6.73 (dd, 1H, J = 0.9 Hz, 3.3 Hz, 30-H), 6.59
(dd, 1H, J = 1.8 Hz, 3.3 Hz, 40-H), 6.54 (dd, 1H, J = 1.8 Hz, 3.3 Hz,
40-H), 6.45 (d, 1H, J = 8.4 Hz, NH), 4.29 (q, 2H, J = 7.2 Hz, OCH2),
3.82–3.75 (m, 1H, CH), 2.90–2.89 (m, 6H, N–CH2), 1.84–1.70 (m,
4H, CH2–CH2), 1.42 (d, 3H, J = 6.3 Hz, –CH–CH3), 1.35 (t, 3H,
J = 6.9 Hz, –OCH2–CH3), 1.27 (t, 6H, J = 6.0 Hz, N–CH2–CH3). MS
(ES) m/z (M+H)+ 507. HRMS calcd for C27H34N6O4 (M+H)+
507.27143; found 507.27187.
Purity: 96.2%) was obtained as a yellow solid. mp 55–60 °C. TLC
(10% MeOH–CHCl3–1% NH4OH): Rf 0.43. 1H NMR (300 MHz, CDCl3):
d 7.55 (bs, 1H, NH), 7.39 (s, 1H, 6-H), 6.38 (d, 1H, J = 8.1 Hz, NH),
4.27 (q, 2H, J = 7.2 Hz, OCH2), 3.83–3.74 (m, 1H, CH), 3.02–2.83
(m, 10H, N–CH2, 2-CH2, 3-CH2), 1.98–1.67 (m, 4H, CH2–CH2),
1.44–1.23 (m, 18H, CH3). MS (ES) m/z(M+H)+ 431. HRMS calcd for
C
23H38N6O2 (M+H)+ 431.31290; found 431.31321.
4.10. Ethyl (8-((4-(diethylamino)butyl)amino)-2,3-
diphenylpyrido[2,3-b]pyrazin-6-yl)carbamate (17)
4.6. Ethyl (8-((5-(diethylamino)pentan-2-yl)amino)-2,3-
di(thiophen-2-yl)pyrido[2,3-b]pyrazin-6-yl)carbamate (7)
The procedure was followed as detailed above using benzil
(57 mg, 0.27 mmol) and the diamine hydrochloride 14 (92 mg,
0.24 mmol). The title compound 17 (30 mg, 24%, HPLC Purity:
100%) was obtained as a yellow solid. mp 155–158 °C. TLC (10%
MeOH–CHCl3–1% NH4OH): Rf 0.27. 1H NMR (300 MHz, CDCl3): d
7.57–7.28 (m, 12H, Ph-H, NH, 6-H), 6.57 (t, 1H, J = 5.4 Hz, NH),
4.31 (q, 2H, J = 6.9 Hz, OCH2) 3.51–3.46 (m, 2H, NH–CH2), 3.00–
2.71 (m, 6H, N–CH2), 1.78–1.78 (m, 4H, CH2–CH2), 1.38 (t, 3H,
J = 6.9 Hz, –OCH2–CH3), 1.23 (t, 6H, J = 6.9 Hz, N–CH2–CH3). MS
(ES) m/z (M+H)+ 513. HRMS calcd for C30H36N6O2 (M+H)+
513.29725; found 513.29753.
The procedure above was followed using 2,20-thenil (95 mg,
0.42 mmol) and the diamine hydrochloride 5 (151 mg, 0.38 mmol).
The title compound 7 (108 mg, 52%, HPLC Purity: 95.4%) was ob-
tained as a yellow solid. mp 78–84 °C. TLC (10% MeOH–CHCl3–1%
NH4OH): Rf 0.47. 1H NMR (300 MHz, CDCl3): d 7.60 (bs, 1H, NH),
7.50 (dd, 1H, J = 1.2 Hz, 5.1 Hz, 50-H), 7.48 (dd, 1H, J = 1.2 Hz,
5.1 Hz, 50-H), 7.44 (s, 1H, 6-H), 7.29–7.24 (dde, 2H, 30-H), 7.09
(dd, 1H, J = 3.9 Hz, 5.1 Hz, 40-H), 6.99 (dd, 1H, J = 3.9 Hz, 5.1 Hz,
40-H), 6.35 (d, 1H, J = 8.4 Hz, NH), 4.25 (q, 2H, J = 6.9 Hz, OCH2),
3.79–3.76 (m, 1H, CH), 2.87–2.80 (m, 6H, N–CH2), 1.80–1.71 (m,
4H, CH2–CH2), 1.39 (d, 3H, J = 6.9 Hz, –CH–CH3), 1.35 (t, 3H,
J = 7.2 Hz, –OCH2–CH3), 1.22 (t, 6H, J = 6.3 Hz, N–CH2–CH3). MS
(ES) m/z (M+H)+ 539. HRMS calcd for C27H34N6O2S2 (M+H)+
539.22574; found 539.22571.
4.11. Ethyl (8-((4-(diethylamino)butyl)amino)-2,3-di(thiophen-
2-yl)pyrido[2,3-b]pyrazin-6-yl)carbamate (18)
The procedure was followed as detailed above using 2,20-thenil
(202 mg, 0.91 mmol) and the diamine hydrochloride 14 (257 mg,
0.69 mmol). The title compound 18 (170 mg, 47%, HPLC Purity:
91.9%) was obtained as a yellow solid. mp 125–129 °C. TLC (10%
MeOH–CHCl3–1% NH4OH): Rf 0.16. 1H NMR (300 MHz, CDCl3): d
7.60 (bs, 1H, NH), 7.49 (dd, 1H, J = 1.2 Hz, 5.1 Hz, 50-H), 7.47 (dd,
1H, J = 1.2 Hz, 5.1 Hz, 50-H), 7.44 (s, 1H, 6-H), 7.27–7.24 (m, 2H,
30-H), 7.08 (dd, 1H, J = 3.6 Hz, 5.1 Hz, 40-H), 6.99 (dd, 1H, 3.9 Hz,
5.1 Hz, 40-H), 6.61 (t, 1H, J = 5.4 Hz, NH), 4.30 (q, 2H, J = 6.9 Hz,
OCH2), 3.47 (dd, 2H, J = 6.3 Hz, 12.6 Hz, NH–CH2), 2.75–2.59 (m,
6H, N–CH2), 1.81–1.69 (m, 4H, CH2–CH2), 1.37 (t, 3H, 7.2 Hz, –
OCH2–CH3), 1.12 (t, 6H, J = 6.3 Hz, N–CH2–CH3). MS (ES) m/z
(M+H)+ 525. HRMS calcd for C26H32N6O2S2 (M+H)+ 525.21009;
found 525.20959.
4.7. Ethyl (8-((5-(diethylamino)pentan-2-yl)amino)-2,3-
di(pyridin-2-yl)pyrido[2,3-b]pyrazin-6-yl)carbamate (8)
The procedure was followed as detailed above using 2,20-pyridil
(106 mg, 0.5 mmol) and the diamine hydrochloride 5 (200 mg,
0.5 mmol). The title compound 8 (90 mg, 33%, HPLC Purity:
95.0%) was obtained as a yellow solid. mp 99–104 °C. TLC (10%
MeOH–CHCl3–1% NH4OH): Rf 0.6. 1H NMR (300 MHz, CDCl3): d
8.37–8.35 (m, 1H, 60-H), 8.26–8.23 (m, 1H, 60-H), 8.21 (d, 1H,
J = 7.8 Hz, 30-H), 7.89–7.80 (m, 3H, 30-H, 50-H), 7.58 (bs, 1H, NH),
7.52 (s, 1H, 6-H), 7.25–7.19 (m, 2H, 40-H), 6.61 (d, 1H, J = 8.1 Hz,
NH), 4.29 (q, 2H, J = 6.9 Hz, OCH2), 3.89–3.80 (m, 1H, CH), 3.16–
2.94 (m, 6H, N–CH2), 2.03–1.91 (m, 2H, CH2), 1.89–1.71 (m, 2H,
CH2), 1.45–1.27 (m, 12H, CH3). MS (ES) m/z (M+H)+ 529. HRMS
calcd for C29H36N8O2 (M+H)+ 529.30340; found 529.30355.
4.12. Ethyl (8-((4-(diethylamino)butyl)amino)-2,3-di(furan-2-
yl)pyrido[2,3-b]pyrazin-6-yl)carbamate (19)
The procedure was followed as detailed above using furil
(172 mg, 0.91 mmol) and the diamine hydrochloride 14
(257 mg, 0.69 mmol). The title compound 19 (120 mg, 36%, HPLC
Purity: 100%) was obtained as a yellow solid. mp 125–129 °C. TLC
(10% MeOH–CHCl3–1% NH4OH): Rf 0.42. 1H NMR (300 MHz,
MeOH-d4): d 7.80 (d, 1H, J = 1.5 Hz, 50-H), 7.74 (d, 1H, J = 1.2 Hz,
50-H), 7.07 (d, 1H, J = 3.0 Hz, 30-H), 6.92 (d, 1H, J = 0.6 Hz, 30-H),
6.71- 6.68 (m, 2H, 40-H), 6.61 (bs, 1H, 6-H), 4.44 (q, 2H,
J = 6.3 Hz, OCH2), 3.62 (bt, 2H, NH–CH2), 3.27–3.18 (m, 6H,
NCH2), 1.87–1.86 (m, 4H, CH2–CH2), 1.43 (t, 3H, J = 6.9 Hz,
OCH2–CH3), 1.34 (t, 6H, J = 7.2 Hz, N–CH2–CH3). MS (ES) m/z
(M+H)+ 493. HRMS calcd for C26H32N6O4 (M+H)+ 493.25578;
found 493.25638.
4.8. Ethyl (8-((5-(diethylamino)pentan-2-yl)amino)-2,3-
dimethylpyrido[2,3-b]pyrazin-6-yl)carbamate (9)
The procedure was followed as detailed above using 2,3-
butanedione (0.064 mL, 0.73 mmol) and the diamine hydrochlo-
ride 5 (190 mg, 0.48 mmol). The title compound 9 (70 mg, 36%,
HPLC Purity: 100%) was obtained as a yellow solid. mp 181–
184 °C. TLC (10% MeOH–CHCl3–1% NH4OH): Rf 0.3. 1H NMR
(300 MHz, CDCl3): d 7.60 (bs, 1H, NH), 7.39 (s, 1H, 6-H), 6.38 (d,
1H, J = 8.4 Hz, NH), 4.24 (q, 2H, J = 6.9 Hz, OCH2), 3.81–3.73 (m,
1H, CH), 3.07–2.85 (m, 6H, N–CH2), 2.68 (s, 3H, 3-CH3), 2.65 (s,
3H, 2-CH3), 1.94–1.67 (m, 4H, CH2–CH2), 1.43–1.28 (m, 12H,
CH3). MS (ES) m/z (M+H)+ 403. HRMS calcd for C21H34N6O2
(M+H)+ 403.28160; found 403.28194.
4.13. Ethyl (8-((2-(diethylamino)ethyl)amino)-2,3-
diphenylpyrido[2,3-b]pyrazin-6-yl)carbamate (20)
4.9. Ethyl (8-((5-(diethylamino)pentan-2-yl)amino)-2,3-
diethylpyrido[2,3-b]pyrazin-6-yl)carbamate (10)
The procedure was followed as detailed above using benzil
(100 mg, 0.47 mmol) and the diamine 15 (147 mg, 0.47 mmol).
The title compound 20 (113 mg, 49%, HPLC Purity: 97.1%) was ob-
tained as a yellow solid. mp 158–164 °C. TLC (10% MeOH–CHCl3–
1% NH4OH): Rf 0.75. 1H NMR (300 MHz, CDCl3): d 7.59–7.50 (m,
The procedure was followed as detailed above using 3,4-hex-
anedione (0.05 mL, 0.4 mmol) and the diamine hydrochloride 5
(143 mg, 0.37 mmol). The title compound 10 (80 mg, 51%, HPLC

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B. Mathew et al. / Bioorg. Med. Chem. 19 (2011) 7120–7128
6H, Ph-H), 7.45 (s, 1H, NH), 7.34–7.25 (m, 6H, Ph-H, 6-H, NH), 4.31
(q, 2H, J = 7.2 Hz, OCH2), 3.41 (dd, 2H, J = 6.3 Hz, 11.7 Hz, NH–CH2),
2.84 (t, 2H, J = 6.3 Hz, CH2–CH2–N), 2.64 (q, 4H, J = 6.9 Hz, N–CH2),
1.38 (t, 3H, J = 7.2 Hz, OCH2–CH3), 1.09 (t, 6H, J = 7.2 Hz, N–CH2–
CH3). MS (ES) m/z (M+H)+ 485. HRMS calcd for C28H32N6O2
(M+H)+ 485.26595; found 485.26651.
4.18. Ethyl (8-((2-(dimethylamino)ethyl)amino)-2,3-di(furan-2-
yl)pyrido[2,3-b]pyrazin-6-yl)carbamate (25)
The procedure was followed as detailed above using furil
(136 mg, 0.71 mmol) and the diamine 16 (200 mg, 0.71 mmol).
The title compound 25 (95 mg, 31%, HPLC Purity: 100%) was ob-
tained as a yellow solid. mp 153–156 °C. TLC (10% MeOH–CHCl3–
1% NH4OH): Rf 0.66. 1H NMR (300 MHz, CDCl3): d 7.59 (d, 2H,
J = 18.0 Hz, 50-H), 7.53 (bs, 1H, NH), 7.35 (s, 1H, 6-H), 7.14 (d, 1H,
J = 3.0 Hz, 30-H), 6.80 (d, 1H, J = 3.3 Hz, 30-H), 6.56 (dd, 2H,
J = 1.5 Hz, 12.6 Hz, 40-H), 4.25 (q, 2H, J = 6.9 Hz, OCH2), 3.93 (bs,
2H, NH–CH2), 3.40 (bs, 2H, CH2–CH2–N), 2.91 (s, 6H, NCH3), 1.36
(t, 3H, J = 6.9 Hz, OCH2–CH3). MS (ES) m/z (M+H)+ 437. HRMS calcd
for C22H24N6O4 (M+H)+ 437.19318; found 437.19439.
4.14. Ethyl (8-((2-(diethylamino)ethyl)amino)-2,3-di(thiophen-
2-yl)pyrido[2,3-b]pyrazin-6-yl)carbamate (21)
The procedure was followed as detailed above, using 2,20-thenil
(196 mg, 0.87 mmol) and the diamine 15 (228 mg, 0.73 mmol). The
title compound 21 (250 mg, 69%, HPLC Purity: 100%) was obtained
as a yellow solid. mp 145–148 °C. TLC (10% MeOH–CHCl3–1%
NH4OH): Rf 0.84. 1H NMR (300 MHz, CDCl3): d 7.52–7.46 (m, 5H,
50-H, 30-H, NH, 6-H), 7.38 (d, 1H, J = 3.6 Hz, 30-H,), 7.05 (dd, 1H,
J = 3.6 Hz, 5.1 Hz, 40-H), 7.02 (dd, 1H, J = 3.6 Hz, 5.1 Hz, 40-H), 4.29
(q, 2H, J = 6.9 Hz, OCH2), 4.20–4.05 (m, 2H, NH–CH2), 3.36 (t, 2H,
J = 6.3 Hz, CH2–CH2–N), 3.25 (q, 4H, J = 6.6 Hz, N–CH2), 1.45 (t,
6H, J = 6.9 Hz, N–CH2–CH3), 1.37 (t, 3H, J = 7.2 Hz, OCH2–CH3). MS
(ES) m/z (M+H)+ 497. HRMS calcd for C24H28N6O2S2 (M+H)+
497.17879; found 497.17947.
4.19. N8-(5-(diethylamino)pentan-2-yl)-2,3-di(furan-2-
yl)pyrido[2,3-b]pyrazine-6,8-diamine (26)
To a stirred solution of KOH (51 mg, 0.92 mmol) in EtOH
(10 mL) was added 6 (100 mg, 0.19 mmol) under argon atmo-
sphere. The reaction mixture was stirred at reflux for 3 hrs.
Excess ethanol was removed under reduced pressure. The dry
residue was dissolved in 50% NaOH solution (10 mL) and
extracted with diethyl ether (3 ꢀ 25 mL). The combined organic
layer was dried over anhydrous sodium sulfate, filtered and con-
centrated in vacuo to afford 26 (25 mg, 29%, HPLC Purity: 100%)
as a yellow solid. mp 125–127 °C. TLC (10% MeOH–CHCl3–1%
NH4OH): Rf 0.13. 1H NMR (300 MHz, CDCl3): d 7.54 (dd, 1H,
J = 0.6 Hz, 1.8 Hz, 50-H), 7.48 (dd, 1H, J = 0.9 Hz, 1.8 Hz, 50-H), 6.88
(dd, 1H, J = 0.9 Hz, 3.6 Hz, 30-H), 6.61 (dd, 1H, J = 0.9 Hz, 3.6 Hz,
30-H), 6.55 (dd, 1H, J = 1.8 Hz, 3.3 Hz, 40-H), 6.51 (dd, 1H,
J = 1.8 Hz, 3.3 Hz, 40-H), 6.22 (d, 1H, J = 8.4 Hz, NH), 5.79 (s, 1H, 6-
H), 4.89 (s, 2H, NH2), 3.64–3.55 (m, 1H, CH), 2.58–2.41 (m, 6H,
N–CH2), 1.71–1.55 (m, 4H, CH2–CH2), 1.33 (d, 3H, J = 6.3 Hz, CH–
CH3), 1.03 (t, 6H, J = 7.2 Hz, N–CH2–CH3). MS (ES) m/z (M+H)+
435. HRMS calcd for C24H30N6O2 (M+H)+ 435.25030; found
435.25054.
4.15. Ethyl (8-((2-(diethylamino)ethyl)amino)-2,3-di(furan-2-
yl)pyrido[2,3-b]pyrazin-6-yl)carbamate (22)
The procedure was followed as detailed above using furil
(300 mg, 1.6 mmol) and the diamine 15 (400 mg, 1.3 mmol).
The title compound 22 (320 mg, 54%, HPLC Purity: 100%) was ob-
tained as a yellow solid. mp 160–162 °C. TLC (10% MeOH–CHCl3–
1% NH4OH): Rf 0.74. 1H NMR (300 MHz, CDCl3): d 8.33 (bs, 1H,
NH), 7.61–7.34 (m, 4H, 50-H, 30-H, 6-H), 6.89 (bs, 1H, 30-H),
6.61–6.57 (m, 2H, 40-H), 4.40–4.13 (m, 4H, OCH2, NH–CH2),
3.40–3.20 (m, 6H, NCH2), 1.52–1.37 (m, 9H, CH3). MS (ES) m/z
(M+H)+ 465. HRMS calcd for C24H28N6O4 (M+H)+ 465.22448;
found 465.22478.
4.16. Ethyl (8-((2-(dimethylamino)ethyl)amino)-2,3-
diphenylpyrido[2,3-b]pyrazin-6-yl)carbamate (23)
4.20. N8-(5-(diethylamino)pentan-2-yl)-2,3-di(thiophen-2-
yl)pyrido[2,3-b]pyrazine-6,8-diamine (27)
The procedure was followed as detailed above using benzil
(403 mg, 1.9 mmol) and the diamine 16 (446 mg, 1.58 mmol).
The title compound 23 (260 mg, 36%, HPLC Purity: 100%) was ob-
tained as a yellow solid. mp 203–205 °C. TLC (10% MeOH–CHCl3–
1% NH4OH): Rf 0.7. 1H NMR (300 MHz, DMSO-d6): d 10.63 (bs,
1H, NH), 8.75 (bs, 1H, NH), 7.59–7.37 (m, 10H, Ph-H), 7.28 (s, 1H,
6-H), 4.32 (q, 2H, J = 6.6 Hz, OCH2), 3.87 (t, 2H, J = 6.9 Hz, NH–
CH2), 3.46 (t, 2H, J = 4.8 Hz, CH2–CH2–N), 2.89 (s, 6H, NCH3), 1.33
(t, 3H, J = 7.2 Hz, OCH2–CH3). MS (ES) m/z (M+H)+ 457. HRMS calcd
for C26H28N6O2 (M+H)+ 457.23465; found 457.23562.
The procedure was followed as detailed above using 7 (185 mg,
0.34 mmol) and KOH (90 mg, 1.6 mmol). The title compound 27
(65 mg, 41%, HPLC Purity: 95.3%) was obtained as a yellow solid.
mp 45–47 °C. TLC (10% MeOH–CHCl3–1% NH4OH); Rf 0.15. 1H
NMR (300 MHz, CDCl3): d 7.46 (dd, 1H, J = 1.2 Hz, 5.1 Hz, 50-H),
7.43 (dd, 1H, J = 1.2 Hz, 5.1 Hz, 50-H), 7.22 (dd, 1H, J = 1.2 Hz,
3.6 Hz, 30-H), 7.20 (dd, 1H, J = 1.2 Hz, 3.9 Hz, 30-H), 7.07 (dd, 1H,
J = 3.6 Hz, 5.1 Hz, 40-H), 6.96 (dd, 1H, J = 3.6 Hz, 5.1 Hz, 40-H), 6.16
(d, 1H, J = 8.4 Hz, NH), 5.77 (s, 1H, 6-H), 4.90 (s, 2H, NH2), 3.64–
3.56 (m, 1H, CH), 2.55–2.42 (m, 6H, N–CH2), 1.69–1.53 (m, 4H,
CH2–CH2), 1.31 (d, 3H, J = 6.3 Hz, CH–CH3), 1.023 (t, 6H, J = 6.9 Hz,
N–CH2–CH3). MS (ES) m/z (M+H)+ 467. HRMS calcd for
4.17. Ethyl (8-((2-(dimethylamino)ethyl)amino)-2,3-
di(thiophen-2-yl)pyrido[2,3-b]pyrazin-6-yl)carbamate (24)
C
24H30N6S2 (M+H)+ 467.20461; found 467.20412.
The procedure was followed as detailed above using 2,20-thenil
(157 mg, 0.71 mmol) and the diamine 16 (200 mg, 0.71 mmol). The
title compound 24 (200 mg, 60%, HPLC Purity: 96.3%) was obtained
as a yellow solid. mp 120–125 °C. TLC (10% MeOH–CHCl3–1%
NH4OH): Rf 0.72. 1H NMR (300 MHz, CDCl3): d 7.58 (s, 1H, NH),
7.48–7.46 (m, 2H, 50-H), 7.42 (s, 1H, 6-H), 7.30–7.27 (m, 2H, 30-
H), 7.06 (dd, 1H, J = 3.6 Hz, 5.1 Hz, 40-H), 7.00 (dd, 1H, J = 3.6 Hz,
5.1 Hz, 40-H), 6.95 (t, 1H, J = 5.4 Hz, NH), 4.30 (q, 2H, J = 6.9 Hz,
OCH2), 3.47 (dd, 2H, J = 6.0 Hz, 11.7 Hz, NH–CH2), 2.70 (t, 2H,
J = 6.0 Hz, CH2–CH2–N), 2.33 (s, 6H, NCH3), 1.37 (t, 3H, J = 6.9 Hz,
OCH2–CH3). MS (ES) m/z (M+H)+ 469. HRMS calcd for C22H24N6O2S2
(M+H)+ 469.14749; found 469.14839.
4.21. Ethyl (4-chloro-6-((5-(diethylamino)pentan-2-
yl)amino)pyrimidin-2-yl)carbamate (29)
To a stirred solution of 4,6-dichloro-2-pyrimidinyl carbamic
acid ethyl ester, 28 (1.0 g, 4.2 mmol) in dry methanol (20 mL), 2-
amino-5-diethylaminopentane (0.84 mL, 4.2 mmol) was added.
The mixture was refluxed for 3 h under nitrogen atmosphere. Ex-
cess methanol was evaporated under reduced pressure and the
resulting residue was purified by silica gel column chromatogra-
phy (3% MeOH–CHCl3–1% NH4OH) to afford 29 (1.22 g, 80%) as a
white foam. TLC (10% MeOH–CHCl3): Rf 0.32. 1H NMR (300 MHz,

B. Mathew et al. / Bioorg. Med. Chem. 19 (2011) 7120–7128
7127
CDCl3): d 6.20–6.15 (m, 2H, NH, 5-H), 4.24–4.17 (m, 3H, OCH2, CH),
3.15–2.82 (m, 6H, NCH2), 2.02–1.56 (m, 4H, CH2–CH2), 1.43–1.20
(m, 12H, CH3). MS (ES) m/z (M+H)+ 357. HRMS calcd for
CH2), 1.32–1.23 (m, 12H, CH3). MS (ES) m/z (M+H)+ 401. HRMS
calcd for C16H28N6O4S (M+H)+ 401.19655; found 401.19751.
C
16H28ClN5O2 (M+H)+ 358.2004; found 358.2008.
4.26. Ethyl (4-((4-(diethylamino)butyl)amino)-6-mercapto-5-
nitropyrimidin-2-yl)carbamate (34)
4.22. Ethyl (4-chloro-6-((4-
(diethylamino)butyl)amino)pyrimidin-2-yl)carbamate (30)
The procedure was followed as detailed above using 32 (600 mg,
1.54 mmol) and potassium thioacetate (264 mg, 2.3 mmol). The ti-
tle compound 34 (80 mg, 13%) was obtained as a syrup. TLC (10%
MeOH–CHCl3–1% NH4OH): Rf 0.21. MS (ES) m/z (M+H)+ 387.
The procedure was followed as detailed above using 4-diethy-
laminobutylamine (447 mg, 3.1 mmol) and 4,6-dichloro-2-pyri-
midinyl carbamic acid ethyl ester, 28 (0.75 g, 3.1 mmol). The title
compound 30 (1.0 g, 92%) was obtained as a syrup. TLC (10%
MeOH–CHCl3): Rf 0.17. 1H NMR (300 MHz, CDCl3): d 6.00 (s, 1H,
5-H), 4.23 (q, 2H, J = 6.9 Hz, OCH2), 3.25 (bs, 2H, NH–CH2), 2.59
(q, 4H, J = 6.0 Hz, N–CH2–CH3), 2.48 (t, 2H, J = 6.9 Hz, CH2–CH2–
N), 1.68–1.50 (m, 4H, CH2–CH2), 1.32 (t, 3H, J = 7.2 Hz, OCH2–
CH3), 1.07 (t, 6H, J = 7.2 Hz, N–CH2–CH3). MS (ES) m/z (M+H)+ 344.
4.27. Ethyl (5-amino-4-((5-(diethylamino)pentan-2-yl)amino)-
6-mercaptopyrimidin-2-yl)carbamate (35)
To a stirred solution of 33 (80 mg, 0.2 mmol) in acetic acid
(5 mL) was added zinc dust (0.4 g) portion wise over a period of
30 minutes at 80 °C under N2 atmosphere. The reaction mixture
was cooled, and the insoluble material was removed by filtration
and washed with acetic acid (1 mL). The filtrate and wash were
combined and evaporated to dryness, and the resulting brownish
oil was triturated with an aqueous solution of 0.1 M K2HPO4
(3 mL). The mixture was extracted with chloroform (3 ꢀ 20 mL).
The combined organic layer was separated, collected, dried over
anhydrous sodium sulfate, filtered and concentrated in vacuo to
give 35 (60 mg, 81%).TLC (10% MeOH–CHCl3–1% NH4OH): Rf 0.25.
1H NMR (300 MHz, CDCl3): d 4.27–4.20 (m, 3H, CH, OCH2), 2.57–
2.35 (m, 6H, NCH2), 1.52–1.47 (m, 4H, CH2–CH2), 1.33 (t, 3H,
J = 7.2 Hz, OCH2–CH3), 1.20 (d, 3H, J = 6.6 Hz, CH–CH3), 1.02 (t,
6H, J = 6.9 Hz, N–CH2–CH3). MS (ES) m/z (M+H)+ 370.
4.23. Ethyl (4-chloro-6-((5-(diethylamino)pentan-2-yl)amino)-
5-nitropyrimidin-2-yl)carbamate (31)
To a stirred solution of 29 (0.32 g, 0.89 mmol) in 1.12 mL of con-
centrated H2SO4, 0.22 mL of fuming nitric acid (sp. gr 1.49) was
added drop wise. The temperature during the addition was kept
between 30–35 °C. After addition, the solution was stirred at 30–
35 °C for 35 min. The mixture was added to crushed ice with vigor-
ous stirring. The pH was adjusted to 8–9 by dropwise addition of
concentrated aqueous ammonia, keeping the temperature below
15 °C. The product separated as an oil, and the mixture was ex-
tracted with chloroform (3 ꢀ 25 mL). The organic extracts were
dried over anhydrous sodium sulfate, filtered, concentrated in va-
cuo providing 0.2 g of the crude mixture. The mixture was purified
over a short pad of silica gel using 2% MeOH–CHCl3–1% NH4OH.
The fractions containing pure compound were concentrated in
vacuo to give pure 31 (144 mg, 40%). TLC (10% MeOH–CHCl3–1%
NH4OH): Rf 0.41. 1H NMR (300 MHz, CDCl3): d 7.93 (d, 1H,
J = 8.1 Hz, NH), 4.50–4.40 (m, 1H, CH), 4.25 (q, 2H, J = 6.9 Hz,
OCH2), 2.75–2.45 (m, 6H, NCH2), 1.70–1.58 (m, 4H, CH2–CH2),
1.35–1.29 (m, 6H, OCH2–CH3, CH–CH3), 1.07 (t, 6H, J = 7.2 Hz, N–
CH2–CH3). MS (ES) m/z (M+H)+ 403. HRMS calcd for C16H27ClN6O4
(M+H)+ 403.18551; found 403.18595.
4.28. Ethyl (5-amino-4-((4-(diethylamino)butyl)amino)-6-
mercaptopyrimidin-2-yl)carbamate (36)
The procedure was followed as detailed above using 34 (80 mg,
0.2 mmol). The title compound 36 was obtained in 55% (40 mg)
yield. TLC (10% MeOH–CHCl3–1% NH4OH): Rf 0.07. 1H NMR
(300 MHz, CDCl3): d 5.84 (bs, 1H, NH), 4.34 (q, 2H, J = 6.9 Hz,
OCH2), 3.39 (m, 2H, NH–CH2), 2.61–2.47 (m, 6H, NCH2), 1.60–
1.54 (m, 4H, CH2–CH2), 1.36 (t, 3H, J = 6.9 Hz, OCH2–CH3), 1.04 (t,
6H, J = 7.2 Hz, N–CH2–CH3). MS (ES) m/z (M+H)+ 357.
4.29. Ethyl (4-((5-(diethylamino)pentan-2-yl)amino)-6,7-
diphenyl-7H-pyrimido[4,5-b][1,4]thiazin-2-yl) carbamate (37)
4.24. Ethyl (4-chloro-6-((4-(diethylamino)butyl)amino)-5-
nitropyrimidin-2-yl)carbamate (32)
To a solution of 35 (50 mg, 0.135 mmol) and sodium acetate
(55 mg, 0.67 mmol) in water–ethanol (1 mL, 1:1) was added desyl
chloride (946 mg, 0.2 mmol). The reaction mixture was stirred at
room temperature for 4 h. The solvent was evaporated in vacuo
and flash column chromatography of the residue (7% MeOH–
CHCl3) yielded 37 (56 mg, 77%, HPLC Purity: 95.6%) as a mixture
of diastereomers. mp 130–135 °C. TLC (10% MeOH–CHCl3–1%
NH4OH): Rf 0.43. 1H NMR (300 MHz, CDCl3): d 7.89–7.85 (m, 2H,
Ph-H), 7.45–7.38 (m, 3H, Ph-H, NH), 7.24–7.15 (m, 6H, Ph-H),
6.15–6.09 (m, 1H, NH), 5.39 (2s, 1H, S–CH–Ph), 4.28–4.27 (m, 1H,
CH), 4.23 (q, 2H, J = 7.2 Hz, OCH2), 2.58–2.46 (m, 6H, NCH2),
1.62–1.55 (m, 4H, CH2–CH2), 1.32–1.25 (m, 6H, CH–CH3, OCH2–
CH3), 1.05–0.95 (m, 6H, N–CH2–CH3). MS (ES) m/z (M+H)+ 547.
HRMS calcd for C30H38N6O2S (M+H)+ 547.28497; found 547.28484.
The procedure was followed as detailed above using 30
(640 mg, 1.86 mmol). The title compound 32 (600 mg, 83%) was
obtained as a syrup. TLC (10% MeOH–CHCl3–1% NH4OH): Rf 0.26.
MS (ES) m/z (M+H)+ 389.
4.25. Ethyl (4-((5-(diethylamino)pentan-2-yl)amino)-6-
mercapto-5-nitropyrimidin-2-yl)carbamate (33)
A solution of 31 (160 mg, 0.39 mmol) and potassium thioace-
tate (66.8 mg, 0.585 mmol) in ethanol (5 mL) was refluxed for
2 h. Excess solvent was removed and the residue was dissolved
in chloroform. The solution was washed with water, and the com-
bined organic layers were dried over anhydrous sodium sulfate,
filtered, concentrated and purified over a short pad of silica gel
(70–230 mesh) using 3.5% MeOH–CHCl3–1% NH4OH. Selected frac-
tions were collected and concentrated in vacuo to give pure 33
(80 mg, 50%). TLC (10% MeOH–CHCl3–1% NH4OH): Rf 0.14. 1H
NMR (300 MHz, CDCl3): d 9.46 (d, 1H, J = 7.5 Hz, NH), 4.24–4.19
(m, 3H, CH, OCH2), 3.35–3.20 (m, 1H, NCH2), 3.10–3.00 (m, 2H,
NCH2), 2.90–2.84 (m, 2H, NCH2), 2.72–2.60 (m, 1H, NCH2), 2.00–
1.85 (m, 1H, CH2), 1.84–1.65 (m, 2H, CH2), 1.55–1.40 (m, 1H,
4.30. Ethyl (4-((4-(diethylamino)butyl)amino)-6,7-diphenyl-7H-
pyrimido[4,5-b][1,4]thiazin-2-yl)carbamate (38)
The procedure was followed as detailed above using 36 (40 mg,
0.11 mmol). The title compound 38 (50 mg, 83%, HPLC Purity:
96.6%) was obtained as a yellow solid. mp 135–140 °C. TLC (10%
MeOH–CHCl3–1% NH4OH): Rf 0.31. 1H NMR (300 MHz, CDCl3): d

7128
B. Mathew et al. / Bioorg. Med. Chem. 19 (2011) 7120–7128
7. Huang, Q.; Kirikae, F.; Kirikae, T.; Pepe, A.; Amin, A.; Respicio, L.; Slaydon, R. A.;
Tonge, P. J.; Ojima, I. J. Med. Chem. 2006, 49, 463.
8. Haydon, D. J.; Stokes, N. R.; Ure, R.; Galbraith, G.; Bennett, J. M.; Brown, D. R.;
Baker, P. J.; Barynin, V. V.; Rice, D. W.; Sedelnikova, S. E.; Heal, J. R.; Sheridan, J.
M.; Aiwale, S. T.; Chauhan, P. K.; Srivastava, A.; Taneja, A.; Collins, I.; Errington,
J.; Czaplewski, L. G. Science 2008, 321, 1673.
9. Kumar, K.; Awasthi, D.; Lee, S.-Y.; Zanardi, I.; Ruzsicska, B.; Knudson, S.; Tonge,
P. J.; Slayden, R. A.; Ojima, I. J. Med. Chem. 2011, 54, 374.
10. Kumar, K.; Awasthi, D.; Berger, W. T.; Tonge, P. J.; Slayden, R. A.; Ojima, I. Future
Med. Chem. 2010, 2, 1305.
7.90–7.87 (m, 2H, Ph-H), 7.44–7.38 (m, 2H, Ph-H), 7.25–7.17 (m,
6H, Ph-H), 7.15 (s, 1H, NH), 6.38 (t, 1H, J = 5.7 Hz, NH), 5.40 (s,
1H, S–CH–Ph), 4.23 (q, 2H, J = 6.9 Hz, OCH2), 3.63–3.52 (m, 2H,
NH–CH2), 2.56–2.48 (m, 6H, NCH2), 1.73–1.66 (m, 4H, CH2–CH2),
1.30 (t, 3H, J = 7.2 Hz, OCH2–CH3), 1.04 (t, 6H, J = 6.9 Hz, N–CH2–
CH3). MS (ES) m/z (M+H)+ 533. HRMS calcd for C29H36N6O2S
(M+H)+ 533.26932; found 533.26932.
11. White, E. L.; Suling, W. J.; Ross, L. J.; Seitz, L. E.; Reynolds, R. C. J. Antimicrob.
Chemother. 2002, 50, 111.
Acknowledgements
12. Reynolds, R. C.; Srivastava, S.; Ross, L. J.; Suling, W. J.; White, E. L. Bioorg. Med.
Chem. Lett. 2004, 14, 3161.
13. Temple, C.; Rose, J. D.; Elliot, R. D.; Montgomery, J. A. J. Med. Chem. 1968, 11,
1216.
14. Elliot, R. D.; Temple, C.; Montgomery, J. A. J. Org. Chem. 1966, 31, 1890.
15. Boldyrev, I. V.; Valdimirtsev, I. F.; Romanenko, E. A.; Korzhe-Nevskaya, N. G.;
Titov, E. V.; Cherkasov, V. M. Chem. Heterocycl. Compd. USSR 1977, 1006.
16. O’Brien, D. E.; Cheng, C. C.; Pfleiderer, W. J. J. Med. Chem. 1966, 9, 573.
17. Temple, C.; Wheeler, G. P.; Comber, R. N.; Elliot, R. D.; Montgomery, J. A. J. Med.
Chem. 1983, 26, 1614.
This work was supported by grants from the National Institutes
of Health 1R01AI50470 and TB contract NO1 Al-95385 through Dr.
Anne Lenaerts at Colorado State University (NIAID Project – Dr.
Robert Goldman).
References and notes
18. Ananthan, S.; Faaleolea, E. R.; Goldman, R. C.; Hobrath, J. V.; Kwong, C. D.;
Laughon, B. E.; Maddry, J. A.; Mehta, A.; Rasmussen, L.; Reynolds, R. C.; Secrist, J.
A.; Shindo, N.; Showe, D. N.; Sosa, M. I.; Suling, W. J.; White, E. L. Tuberculosis
2009, 89, 334.
19. Temple, C. G. Chemistry & Biology of Pteridines; Walter de Gruyter & Co.: New
York, 1990. pp 1009–1014.
20. Temple, C.; Rener, G. A. J. Med. Chem. 1992, 35, 4809.
21. Lenaerts, A. J.; Gruppo, V.; Brooks, J. V.; Orme, I. M. Antimicrob. Agents
Chemother. 2003, 47, 783.
1. Goldman, R. C.; Laughon, B. E. Tuberculosis (Edinb) 2009, 89, 331.
2. Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. Nature 2011, 469, 483.
3. Erickson, H. P. Cell 1995, 80, 367.
4. Wang, J.; Galgoci, A.; Kodali, S.; Hearth, K. B.; Jayasuriya, H.; Dorso, K.; Vicente,
F.; Gonzalez, A.; Cully, D.; Bramhill, D.; Singh, S. J. Biol. Chem. 2003, 278, 44424.
5. Margailt, D. N.; Romberg, L.; Mets, R. B.; Hebert, A. M.; Mitchinson, T. J.;
Krischner, M. W.; Chaudhuri, D. R. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11821.
6. Urgaonkar, G.; Pierre, S. L. H.; Meir, I.; Lund, H.; Chaudhuri, D. R.; Shaw, J. T. Org.
Lett. 2005, 7, 5609.