Investigation of 4-amino-5-alkynylpyrimidine-2(1H)-ones as anti-mycobacterial agents

Article abstract of DOI:10.1016/j.bmc.2016.03.003

In vitro anti-mycobacterial activities of novel 4-amino-5-alkynylpyrimidine-2(1H)-ones were investigated. 4-Amino-5-heptynylpyrimidine-2(1H)-one (3) and 4-amino-5-(2-phenylethynyl)pyrimidine-2(1H)-one (7) displayed potent in vitro activity against Mycobac

Full text of DOI:10.1016/j.bmc.2016.03.003

Accepted Manuscript
Investigation of 4-amino-5-alkynylpyrimidine-2(1H)-ones as anti-mycobacte-
rial agents
Gaurav Garg, Milind Pande, Ambika Agrawal, Jie Li, Rakesh Kumar
PII:
S0968-0896(16)30140-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.03.003
BMC 12852
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 August 2015
23 February 2016
2 March 2016
Please cite this article as: Garg, G., Pande, M., Agrawal, A., Li, J., Kumar, R., Investigation of 4-amino-5-
alkynylpyrimidine-2(1H)-ones as anti-mycobacterial agents, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.03.003
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Investigation of 4-amino-5-alkynylpyrimidine-2(1H)-ones as anti-
mycobacterial agents
Gaurav Garga, Milind Pandeb, Ambika Agrawalc, Jie Lid and Rakesh Kumar*c
aDepartment of Pharmacy, Mangalayatan University, Aligarh, UP, India. b School of
Pharmacy, People’s University, Bhopal, MP, India cDepartment of Laboratory Medicine
and Pathology, dDepartment of Surgery, University of Alberta, Edmonton, AB, Canada T6G
2S2
*Address correspondence to:
728 HMRC, Department of Laboratory Medicine and Pathology
Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
Email: rkumar@ualberta.ca
1

Abstract – In vitro anti-mycobacterial activities of novel 4-amino-5-alkynylpyrimidine-
2(1H)-ones were investigated. 4-Amino-5-heptynylpyrimidine-2(1H)-one (3) and 4-amino-
5-(2-phenylethynyl)pyrimidine-2(1H)-one (7) displayed potent in vitro activity against
Mycobacterium bovis and Mycobacterium tuberculosis. Compounds 3 and 7 were also
assessed for their in vivo activity in BALB/c mice infected with M. tuberculosis (H37Ra).
Both compounds showed promising in vivo efficacy at a dose of 25 mg/kg for 2 weeks.
Importantly, compounds 3 and 7 interacted synergistically with the front-line anti-
tuberculosis drug isoniazid in vitro and in vivo. These results suggest that this class of
compounds has strong anti-mycobacterial potential.
Keywords: Tuberculosis, Heterocycles, Pyrimidines, Anti-mycobacterial agent
2

1. Introduction
Tuberculosis (TB) is one of the deadliest infectious diseases in humans, particularly
in developing countries. Tubercle bacilli spread when a person with active disease creates an
respiratory aerosol by coughing sneezing or talking. Immunocompetent people become
infected by breathing in air that is carrying tubercle bacilli. While anyone can acquire TB
organisms, people with HIV/AIDS, people with diabetic conditions, people undergoing
organ transplantation, the elderly, the homeless, health-professionals and cancer patients are
at high risk.1-5
TB is also a well-recognized public health problem in developed countries such as
the United States of America (USA) and the United Kingdom (UK).6 In Japan, the rate of
TB infection is increasing.7 Despite the presence of effective immunity, co-infection of HIV
with TB facilitates the progression of both HIV and active TB disease to full clinical disease.
TB is the leading cause of opportunistic infection and mortality among HIV infected
persons.8
At the present time, nearly two billion people worldwide are infected with
Mycobacterium tuberculosis (Mtb), and the prevalence of active TB is increasing.1-3,8 The
latent form of infection in many of these individuals may reactivate sometime later in life.
In the presence of effective cellular immunity, tubercle bacilli remain contained and don’t
reproduce or spread. However, if an individual is immunocompromised by human
immunodeficiency virus (HIV) infection, cancer chemotherapy, or use of
immunosuppressive drugs in transplantation, reactivation of the latent TB can lead to full
clinical disease and further dissemination of infectious organism.1-3,8
3

Chemotherapy with anti-tuberculosis drugs such as isoniazid, pyrazinamide
streptomycin, p-amino salicylic acid, and rifampicin revolutionized TB therapy in the 1970s,
resulting in a rapid decline of TB cases in many developed countries. However, current
chemotherapy has become ineffective with the emergence of multi-drug resistant (MDR),
extensively-drug resistance (XDR) and totally-drug resistance (TDR) strains of Mtb.9 The
completely drug-resistance form of disease has been documented in several countries
including India, Italy and Iran.10 To meet the challenges of MDR, XDR and TDR-TB
worldwide, investigation of a new class of anti-mycobacterial agents are of paramount
importance.
The complete sequencing of the Mycobacterium tuberculosis (Mtb) genome has
facilitated the identification of novel targets for developing new anti-tuberculosis drugs.
Several genes have been identified which encode enzymes involved in nucleic acid
synthesis, and pyrimidine and purine biosynthesis.11
DNA polymerase III enzyme is highly conserved in gram-positive bacterial
pathogens with low G+C content. It is absolutely required for their replication, and has been
targeted by 6-anilinouracils (6-AUs).12 6-Anilinouracil derivatives can mimic the guanine
moiety of the purine 2’-deoxyribonucleoside triphosphate dGTP as they form hydrogen
bonds with a cytosine residue in the template strand (Figure 1).13 The aryl group present at
C-6 of 6-AUs selectively binds near the polymerase enzyme active site and thus leads to the
formation of an inactive ternary complex. Mtb DNA sequence analysis has also revealed the
presence of a number of conserved active residues and domains in DNA Pol III.
Importantly, no sequence homology between Mtb DNA pol III and human DNA pol has
been reported.14 In silico screening of a large number of compounds against the modeled
4

Mtb DNA pol III (DnaE2) revealed that 6AUs also have potential as inhibitors of Mtb DNA
polymerase.
Figure 1: Base-pairing of 6-anilinouracils (6-AUs) with cytosine in the DNA template
5-Alkynated nucleosides have contributed significantly in anti-tuberculosis
activities in earlier studies.15 The 5-position of the pyrimidine base in a DNA duplex offers
a site for introducing alkynyl substituents that can protrude in to the major groove and thus
do not sterically interfere with helix formation. And yet they can selectively get incorporated
into the mycobacterial DNA chain and / or inhibit Mtb DNA polymerase.14
Here we report the design, synthesis and evaluation of a new series of 4-amino-5-
alkynylpyrimidine-2(1H)-ones for testing their anti-mycobacterial properties. 4-Amino-5-
alkynylpyrimidine-2(1H)-ones can base-pair with the guanine, component of dGTP by three
cytosine-specific hydrogen bonds, and moieties present at their C-5 position can fit in to the
enzyme binding pocket (Figure 2).14 By simultaneous base-pairing and lipophilic
interactions with enzyme-specific domains, it is possible that the designed pyrimidine
analogs can inhibit the activity of Mtb DNA pol III. Further, a new class of 5-substituted
pyrimidines may be utilized in de novo nucleic acid synthesis by Mtb and get incorporated in
to the growing Mtb DNA chain inhibiting Mtb growth.
5

Figure 2: Base-pairing of the 4-amino-5-alkynyl pyrimidines with guanine in the DNA template
In these studies, we noted that 4-amino-5-heptynyl (3), 4-amino-5-decynyl (4) and
4-amino-5-(2-phenylethynyl) (7) pyrimidines exhibit significant activity against Mtb
(H37Ra) and M.bovis (BCG). Encouragingly, they demonstrated synergistic interactions
when combined with the first-line anti-tuberculosis drug isoniazid. The inhibitors 3 and 7
were evaluated for their efficacy in a murine model of mycobacterial infection alone and in
combination with isoniazid upon oral administration. Intriguingly, compound 3 alone
substantially reduced the Mtb load in lung, liver and spleen tissues while compound 7
exhibited modest inhibition of Mtb in all of the organs. However, compound 7 led to the
increased inhibition of the bacilli in lungs when combined with isoniazid as compared to
compound 3 and isoniazid combined treatment.
2. Chemistry
The novel 4-amino-5-alkynylpyrimidine-2(1H)-ones (3-7) were synthesized in one-
pot reactions by the Pd-catalyzed coupling reaction of 4-amino-5-iodouracil (2) with
respective terminal alkynes using Sonogashira reaction16 to provide 5-heptynyl- (3), 5-
decynyl- (4), 5-tridecynyl- (5), 5-tetradecynyl- (6) and 5-(2-phenylethynyl)- (7) pyrimidines
in 33-67 % yield as depicted in Scheme 1. The starting material 2 was obtained from 5-
6

iodouracil. The compounds (3-7) were >98% pure and their structures were confirmed by 1H
NMR, 13C NMR, FTIR and C, H, N analysis.
2
3-7
Scheme 1. Reagents and conditions: (i) Triisopropyl benzenesulfonyl chloride, 4-
dimethylamino pyridine, triethylamine, acetonitrile, ammonium hydroxide; (ii)
Tetrakis(triphenyl phosphine)palladium, CuI, diisopropyltriethylamine, dimethyl formamide,
room temperature, 1-heptypne (3), 1-decyne (4), 1-tridecyne (5), 1-tetradecyne (6),
phenylacetylene (7).
3. Results and discussion
The anti-mycobacterial activities of the 4-amino-5-alkynylpyrimidine-2(1H)-ones (3-
7) were evaluated against three mycobacteria (M. bovis BCG, M. tuberculosis H37Ra, and
M. avium ATCC 25291) using the microplate Alamar blue assay (MABA) at 1-200 g/mL
concentrations.
To analyze the anti-mycobacterial effect against drug-resistant
mycobacteria, we used rifampicin-resistant strain M. tuberculosis H37Rv (ATCC 35838).
Two drugs in clinical use, rifampicin and clarithromycin were included in these assays as
reference standards. Determination of anti-mycobacterial activity was undertaken as
described in the experimental section. The in vitro test results for compounds 3-7 are
described in Table 1 as MIC50.
7

Table 1: In vitro anti-mycobacterial activity of 4-amino-5-alkynylpyrimidine-2(1H)-ones
against M. bovis, M. tuberculosis and M. avium.
NH2
R
N
O
N
H
3-7
Compound
R
Anti-mycobacterial activitya,b
50% inhibitory concentration (MIC50, g/mL)
M. tuberculosis
(H37Ra)
M. bovis
(BCG)
12.5
M. avium
(ATCC 25291)
0
C
C-(CH2)4CH3
12.5
3
C
C
C
C
C-(CH2)7CH3
C-(CH2)10CH3
C-(CH2)11CH3
C-C6H5
50
0
50
0
0
0
0
0
4
5
6
7
0
0
25
25
Rifampicinc
Clarithromycinc
-
0.5-1 (MIC90)
NDd
1 (MIC90)
ND
2 (MIC90)
2 (MIC90)
-
aAnti-mycobacterial activity was determined at concentrations 200, 100, 50, 25, 12.5, 6.25,
3.25 and 1.56 µg/mL.
bRange from three independent repeated experiments.
cPositive control drugs.
dND = Not determined.
8

Among the 5-alkynylpyrimidine-2(1H)-ones (3-7), a clear structure activity
relationship (SAR) can be delineated. The compounds containing a smaller carbon chain at
the C-2 carbon of the 5-substituent were found to be inhibitors of M. bovis and M.
tuberculosis in these assays, demonstrating activity depending on the length of the alkynyl
side chain. The larger chain containing 5-tridecynyl (5) and tetradecynyl (6) derivatives
were devoid of anti-mycobacterial activity (Table 1). 5-Hepynyl (3) and 5-decynyl (4) side
chains provided activity against both mycobacteria. It was observed that increasing the C4
(heptyne) side chain to C7 (decyne) and further to C10 (tridecyne), significantly reduced
biological activities (Table 1). The inhibition in multiplication of Mtb by the two most
active
analogs,
4-amino-5-heptynylpyrimidin-2(1H)-one
(3)
and
4-amino-5-
decynylpyrimidine-2(1H)-one (4) was: 100% (200,100, 50 g/ml) and 90% (25 g/ml) for 3,
and 100% (200 g/ml) and 50% (50 g/ml) for 4. Inhibition of Mtb by compounds 3 and 4
was similar to that observed for M. bovis. The 4-amino-5-alkynylpyrimidine-2(1H)-ones 3-7
did not exhibit activity against M. avium. Overall, these results suggest that a longer carbon
chain at the C-2 of the 5-position in this series of compounds influences the anti-
mycobacterial activity. Interestingly, 4-amino pyrimidines 3 and 4 led to the inhibition of
mycobacterial replication as compared to their 4-oxo counterparts, which had no activity.17
This suggests that an amino group at C-4 favors anti-mycobacterial activity. Inclusion of the
phenyl ring at the C-2 carbon atom of the 5-substituent in the present series provided a
compound 7 that exhibited superior activity to that of compound 4. This suggests that a
phenyl substituent at the C-2 position of the 5-substituent is acceptable to demonstrate anti-
mycobacterial activity.
9

The most potent inhibitor, 3, was also evaluated for its activity towards drug-
susceptible and rifampicin-resistant strains of M. tuberculosis, H37Rv. Compound 3
demonstrated 50% inhibitory concentration (MIC50) of 12.5 g/mL against both drug-
susceptible and drug-resistant strains. In these assays, rifampicin showed no activity against
the rifampicin-resistant strain at 2.0 g/mL whereas isoniazid provided 100% inhibition of
drug-resistant strain at 1.0 g/mL.
To understand potential and possible interactions of 4-amino-5-alkynylpyrimidine-
2(1H)-ones with current anti-mycobacterial drugs, we selected compounds 3 and 7 to test in
combination with isoniazid to determine their effect on the growth of Mtb (H37Ra) in vitro.
Isoniazid is one of the first-line anti-tuberculosis drug and inhibits mycolic acid synthesis
which is essential for the mycobacterial cell wall formation. However, its monotherapy is
not recommended to treat active tuberculosis due to the development of resistance.18
Pyrimidines 3 and 7 were used at 12.5-0.78 and 25-0.78 g/mL ranges, respectively, and
isoniazid at 0.05 g/mL. It was encouraging to note that both compounds 3 (90% inhibition
at 6.25 g/mL) and 7 (90% inhibition at 6.25 g/mL) provided synergistic inhibition of Mtb
when combined with isoniazid (Figure 3). Isoniazid showed only 10% inhibition at 0.05
g/mL by itself whereas 3 and 7 exhibited no inhibition at 6.25 g/mL. Synergy was
calculated as fractional inhibitory concentration (FIC, quotient), which is the ratio of the
MIC90 of each drug in combination to the MIC90 of that drug when used alone. For a two
drug combination, a sum of quotient (synergy quotient) <0.5 indicates synergistic action, a
sum of quotients of 0.5 - 1 reflects an additive effect, whereas sum of quotients >1 reflects
antagonism.19 The sum of quotients for the combination of compound 3 and isoniazid is
0.37, which is lower than 0.5, suggesting a synergistic interaction. For the combination of 7
10

and isoniazid, the sum of quotients was 0.25, which also reflects synergy. This study
suggests that our new class of pyrimidines is acting at a different target than isoniazid, is not
interacting antagonistically, and would be effective when combined with current drugs.
Figure 3. In vitro combination effect of compound 3 and 7 with isoniazid against M.
tuberculosis (H37Ra).
110
Compound 3 alone
Compound 3+ INH @ 0.05 µg/ml
INH alone @ 0.05 µg/ml
100
90
80
70
60
50
40
30
20
10
0
0
0.78 1.56 3.125 6.25 12.5
25
50
100
Compound 3 concentration (µg/ml)
Compound 7 alone
Compound 7 + INH @ 0.05 µg/ml
INH alone @ 0.05 µg/ml
110
100
90
80
70
60
50
40
30
20
10
0
0
0.78 1.56 3.125 6.25 12.5
25
50
100
Compound 7 concentration (µg/ml)
Synergy quotient of compounds 3 and 7
Compound MIC90 fraction MIC90 fraction Synergy quotient
No.
of the test
compound
of isoniazid
0.12
0.5
0.5
0.37
0.25
3
7
0.006
11

Compounds 3 and 7 were next tested for their efficacy in mice infected with Mtb
(H37Ra) when given orally at a dose of 25 mg/kg, as described in detail in the experimental
section. Isoniazid was used as a reference drug since it exhibited synergy with both
compounds 3 and 7. Notably, compound 3 provided significant reduction in the bacterial
load in lungs, liver, and spleen of the treated animals compared to the untreated control
group (Figure 4). Out of the five mice, compound 3 decreased the bacterial load in lungs,
liver and spleen of all five mice (lungs: 79% inhibition; liver: 50% inhibition; spleen: 51%
inhibition). Reduction in colony forming units (CFUs) by compound 3 was statistically
significant (lungs: P = 0.001; liver: P = 0.05; spleen: P = 0.05) compared to untreated mice.
Compound 3 was found to be much more active than isoniazid administered at 1 mg/kg
dose.
We tested whether the in vitro combinatorial synergistic effect of compound 3 with
first-line drug isoniazid could also be demonstrated in vivo in the mouse model of Mtb
infection. Compound 3 was administered orally at 12.5 and 6.25 mg/kg doses with isoniazid
at 1mg/kg dose. Out of five mice, the combination of 3 at 12.5 mg/kg + isoniazid at 1 mg/kg
decreased Mtb CFUs significantly in the lungs (95-99% inhibition) of two mice, the liver
(96% inhibition) of one mouse, and the spleens (70-75% inhibition) of all five mice. In
contrast, isoniazid alone at 1 mg/kg and compound 3 alone at 12.5 mg/kg (data not shown)
had almost no effect. Intriguingly, when compound 3 was tested at a lower dose of 6.25
mg/kg with isoniazid at 1 mg/kg, we also noted anti-bacterial activity in the lungs (70-78%
inhibition) of two mice and the spleens (40-45% inhibition) of all five mice. Compound 3
alone at 6.25 mg/kg had no activity (data not shown). No antagonism of 3 at 12.5 or 6.25
mg/kg dose with INH was observed. These results confirmed that compound 3 provided
12

better inhibition of Mtb in combination with current drugs where doses of both agents can be
reduced drastically.
Figure 4. Efficacy of compound 3 alone and/or in combination with and isoniazid (INH) in a
murine model of tuberculosis.
Lungs
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Each symbol represents an individual mouse.
13

Figure 5. Efficacy of compound 7 alone and/or in combination with isoniazid (INH) in a
murine model of tuberculosis.
Lungs
20000
***
***
16000
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4000
0
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Each symbol represents an individual mouse.
Compound 7 showed modest or no activity as compared to compound 3 when
administered alone (Figure 5). However, it displayed synergy in combination with isoniazid
14

in in vitro studies. Considering lower efficacy of compound 7 than 3, we tested it in vivo in
combination with isoniazid at a dose of 12.5 mg/Kg only. Interestingly, combination of
compound 7 at a dose of 12.5 mg/kg with isoniazid at 1 mg/kg provided inhibition of Mtb in
the lungs (70-75% inhibition) of all five mice as compared to compound 3 at 12.5 mg/kg +
isoniazid at 1 mg/kg where significant reduction of bacteria was obtained in only two mice.
Compound 7 in combination with isoniazid also led to significant reduction in bacterial
burden in the spleens (45-50% inhibition) of all five mice. This data further suggest that
novel pyrimidines discovered in this program can be combined with first-line anti-
tuberculosis drugs.
Mice administered with compound 3 or 7 alone or in combination with isoniazid for
2 weeks showed no adverse effects in terms of weight loss, behavioral changes, or in the
findings of gross necroscopy after the mice were euthanized. In the serum of treated mice,
selective biochemical parameters (urea, creatinine, phosphate, calcium, cholesterol, alkaline
phosphatase, and amylase) were examined and no significant changes were observed,
suggesting that they do not have kidney or pancreas-related toxicities.
Figure 6. Toxicity of novel compounds 3-7 on VERO cells.
2.5
Compound 3
Compound 4
2.0
Compound 5
Compound 6
1.5
Compound 7
DMSO
1.0
0.5
0.0
0.36 0.78 1.56 3.125 6.25 12.5
25
50
100
200
Concentration (µg/ml)
All data represent mean  S.E.M.
The compounds 3-7 were tested in vitro for their toxicity against a monkey kidney
15

(Vero cells) cell line. No toxicity was observed up to the highest concentrations tested (CC50
= >200g/mL) (Figure 6).
The precise mechanism of action of the active compounds inhibiting mycobacterial
multiplication in this study is not clear. The complete genome sequence of M. tuberculosis
has been deciphered.11 It encodes many of the enzymes required for DNA and RNA
synthesis, and pyrimidine and purine nucleoside biosynthesis. It is postulated that active
compounds may be inhibiting mycobacterial DNA and/or RNA synthesis, by acting as
substrates and/or inhibitors of metabolic enzymes of DNA and/or RNA synthesis, possibly
targeting DNA pol III.
In conclusion, a new class of 4-amino-5-alkynylpyrimidine-2(1H)-ones possessing
5-heptynyl and 5-decynyl and 5-(2-phenylethnyl) substituents were found to be potent and
selective inhibitors of M. tuberculosis and M. bovis multiplication in vitro. This new series
of agents have potential to inhibit drug-resistant Mtb and can be combined with first-line
anti-tuberculosis drugs. Most importantly, oral administration of compounds 3 and 7
afforded efficacy in vivo in a mouse model of tuberculosis without any evidence of toxicity.
New TB drugs are urgently required to shorten the duration of treatment to obtain better
patient compliance, reduce toxicity, and to avoid or delay the emergence of resistance.
4. Experimental
Melting points were determined with a Buchi capillary apparatus and are
1
uncorrected. H NMR and 13C NMR spectra were determined for solutions in Me2SO-d6 on
a Bruker AM 300 spectrometer using Me4Si as an internal standard. 13C NMR (J modulated
spin echo) spectra were determined where methyl and methyne carbon resonances appear as
16

positive peaks, and methylene and quaternary carbon resonances appear as negative peaks.
Chemical shifts are given in ppm relative to TMS as an internal standard. The assignment of
all exchangeable protons (OH, NH) was confirmed by the addition of the D2O. FTIR
spectrum was determined for the most active compounds using KBR pellet. Micro-analyses
were within + 0.4% of theoretical values for all elements listed, unless otherwise indicated.
Silica gel column chromatography was carried out using Merck 7734 silica gel (100-200 M
particle size). Thin layer chromatography (TLC) was performed with Machery-Nagel
Alugram SiL G/uv silica gel slides (20 M thickness). 5-Iodouracil was purchased from
Sigma Aldrich.
4.1. 4-Amino-5-heptynylpyrimidine-2(1H)-one (3)
To a stirred solution of 4-amino-5-iodouracil (200 mg, 0.84 mmol) in anhydrous
dimethylformamide (15 mL) at room temperature in a nitrogen atmosphere were added
tetrakis(triphenylphosphine)palladium (98 mg, 0.08 mmol), copper(I)iodide (32 mg, 0.17
mmol), diisopropylethylamine (0.28 mL, 1.7 mmol) and 1-heptyne (0.33 mL, 2.53 mmol).
The reaction mixture was stirred at room temperature. After 22 h, 10 drops of 5 % of the
disodium salt of EDTA/H2O were added to the reaction mixture, and the mixture was
concentrated in vacuo. The residue obtained was purified on a silica gel column using
MeOH/CHCl3 (4:96, v/v) as an eluent to give 3 (117 mg, 57%) as a solid; mp 225-230° C;
1HNMR (DMSo-d6): δ 0.89 (t, J = 7.14 Hz, 3H, CH3), 1.37-1.24 (m, 4H, 2x CH2), 1.55-1.50
(m, 2H, β-CH2), 2.39 (t, J = 7.2 Hz, 2H, α-CH2), 6.54 (s, 1H, NH2), 7.57 (br s, 1H, NH2),
8.10 (s, 1H, H-6), 10.75 (br s, 1H, NH); 13CNMR (DMSO-d6): δ: 14.27 (CH3), 19.46, 22.10,
28.19, 31.08 (4x CH2), 44.43 (C-β), 72.62 (C-α), 95.54 (C-5), 145.79 (C-6), 155.59 (C-2),
165.92 (C-4). FTIR (KBr) Cm-1: 3475 (Ar C-H), 3387 (NH), 3137 (NH2) 2952, 2932, 2861,
17

2639 (alkyl), 2180 (C C), 1903, 1649 (C=O), 1465, 1295, 1227, 923, 785, 642, 471 Cm-1.
Anal C11H15N3O (M.W. 205.12). Calculated C 64.37, H 7.37, N 20.47. Found C 64.73, H
7.68 N 20.81.
4.2. 4-Amino-5-decynylpyrimidine-2(1H)-one (4)
To a stirred solution of 4-amino-5-iodouracil (200 mg, 0.84 mmol) in anhydrous
dimethylformamide (15 mL) at room temperature in a nitrogen atmosphere were added
tetrakis(triphenylphosphine)palladium (98 mg, 0.08 mmol), copper(I)iodide (32 mg, 0.17
mmol), diisopropylethylamine (0.28mL, 1.7 mmol) and 1-decyne (0.46 mL, 2.53 mmol).
The reaction mixture was stirred at room temperature. After 22 h, 10 drops of 5 % of the
disodium salt of EDTA/H2O were added to the reaction mixture, and the mixture was
concentrated in vacuo. The residue obtained was purified on silica gel column using
MeOH/CHCl3 (4:96, v/v) as an eluent to give 4. This was obtained as a solid in 47% yield;
1
mp 240-245° C; HNMR (DMSO-d6): δ 0.87 (t, J = 6.6 Hz, 3H, CH3), 1.27 (t, J = 1.2 Hz,
8H, 4x CH2), 1.37 (t, J = 6.0 Hz, 2H, γ-CH2), 1.53 (t, J = 7.2 Hz, 2H, β-CH2), 2.39 (t, J = 7.2
Hz, 2H, α-CH2), 6.54 (s, 1H, NH2), 7.51 (br s, 1H, NH2), 7.57 (s, 1H, H-6), 10.75 (br s, 1H,
NH); 13CNMR (DMSO-d6): δ: 14.42 (CH3), 19.50, 22.54, 28.55, 28.86, 28.98, 29.06, 31.72
(7x CH2), 44.41 (C-β), 72.60 (C-α), 95.93 (C-5), 145.74 (C-6), 155.57 (C-2), 165.92 (C-4).
Anal. C14H21N3O (M.W. 247.17). Calculated C 67.98, H 8.56, N 16.99. Found C 67.59, H
8.17, N 16.
4.3. 4-Amino-5-tridecynylpyrimidine-2(1H)-one (5)
To a stirred solution of 4-amino-5-iodouracil (200 mg, 0.84 mmol) in anhydrous
dimethylformamide (15 mL) at room temperature in a nitrogen atmosphere were added
tetrakis (triphenyl phosphine)palladium (98 mg, 0.08 mmol), copper(I)iodide (32 mg, 0.17
18

mmol), diisopropylethylamine (0.28mL, 1.7 mmol) and 1-tridecyne (0.33 mL, 2.53 mmol).
The reaction mixture was stirred at room temperature. After 22 h, 10 drops of 5 % of the
disodium salt of EDTA/H2O were added to the reaction mixture, and the mixture was
concentrated in vacuo. The residue obtained was purified on silica gel column using
MeOH/CHCl3 (4:96, v/v) as an eluent to give 5. This was obtained as a solid in 33% yield;
mp >250° C; 1 HNMR (DMSO-d6): δ 0.86 (t, J = 6.6 Hz, 3H, CH3), 1.25 (d, J = 6.6 Hz, 14H,
7x CH2), 1.36 (t, J = 6.0 Hz, 2H, γ-CH2), 1.52 (t, J = 7.2 Hz, 2H, β-CH2), 2.38 (s, 2H, α-
13
CH2), 6.57 (s, 1H, NH2), 7.47 (br s, 1H, NH2), 7.43 (s, 1H, H-6), 10.83 (br s, 1H, NH). C
NMR (DMSO-d6): δ: 14.40 (CH3), 19.45, 22.53, 28.50, 28.81, 28.97, 29.14, 29.35, 29.42,
29.44, 31.73 (10x CH2), 42.13 (C-β), 63.50 (C-α), 70.23 (C-5), 72.68 (C-6), 70.23 (C-2),
96.07 (C-4). Anal C17H27N3O (M.W. 289.22). Calculated C 70.55, H 9.40, N 14.52. Found
C 70.17, H 9. 78, N 14.90.
4.4. 4-Amino-5-tetradecynylpyrimidine-2(1H)-one (6)
To a stirred solution of 4-amino-5-iodouracil (200 mg, 0.84 mmol) in anhydrous
dimethylformamide (15 mL) at room temperature in a nitrogen atmosphere were added
tetrakis (triphenyl phosphine)palladium (98 mg, 0.08 mmol), copper(I)iodide (32 mg, 0.17
mmol), diisopropylethylamine (0.28mL, 1.7 mmol) and 1-tetradecyne (0.33 mL, 2.53
mmol). The reaction mixture was stirred at room temperature. After 22 h, 10 drops of 5 % of
the disodium salt of EDTA/H2O were added to the reaction mixture, and the mixture was
concentrated in vacuo. The residue obtained was purified on silica gel column using
MeOH/CHCl3 (4:96, v/v) as an eluent to give 6. This was obtained as a solid in 42% yield;
0
mp >250 C; 1 HNMR (DMSO-d6): δ 0.86 (t, J = 6.6 Hz, 3H, CH3), 1.24 (s, 16H, 8x CH2),
1.36 (t, J = 6.0 Hz, 2H, γ-CH2), 1.52 (q, J = 7.2 Hz, H, β-CH2), 2.38 (t, J = 7.2 Hz, 2H, α-
19

13
CH2), 6.56 (s, 1H, NH2), 7.48 (br s, 1H, NH2), 7.42 (s, 1H, H-6), 10.76 (br s, 1H, NH).
CNMR (DMSO-d6): δ: 14.42 (CH3), 19.42, 22.55, 28.59, 28.84, 29.00, 29.16, 29.39, 29.46,
29.49, 31.74, 32.65 (11x CH2), 43.33 (C-β), 63.54 (C-α), 70.42 (C-5), 72.95 (C-6), 70.56 (C-
2), 96.27 (C-4). Anal. C18H29N3O (M.W. 303.45). Calculated C 71.25, H 9.63, N 13.85.
Found C 71.59, H 9.23, N 13.45.
4.5. 4-Amino-5-(2-phenylethynyl)pyrimidine-2(1H)-one (7)
To a stirred solution of 4-amino-5-iodouracil (200 mg, 0.84 mmol) in anhydrous
dimethylformamide (15 mL) at room temperature in a nitrogen atmosphere were added
tetrakis (triphenyl phosphine)palladium (98 mg, 0.08 mmol), copper(I)iodide (32 mg, 0.17
mmol), diisopropylethylamine (0.28mL, 1.7 mmol) and 1-phenylacetylene (0.33 mL, 2.53
mmol). The reaction mixture was stirred at room temperature. After 22 h, 10 drops of 5 % of
the disodium salt of EDTA/H2O were added to the reaction mixture, and the mixture was
concentrated in vacuo. The residue obtained was purified on silica gel column using
MeOH/CHCl3 (4:96, v/v) as an eluent to give 7. This was obtained as a solid in 67% yield;
1
mp >250° C; HNMR (DMSO-d6): δ 6.93 (s, 1H, NH2), 7.40 (m, 3H, C6H5), 7.60 (m, 2H,
C6H5), 7.66 (s, 1H, NH2), 7.83 (s, 1H, H-6), 11.01 (br s, 1H, NH); 13CNMR (DMSO-d6): δ:
56.48 (C-β), 82.30 (C-α), 94.18 (C-5), 128.75, 128.90, 131.61 (C-phenyl), 147.30 (C-6),
155.52 (C-2), 165.30 (C-4). FTIR (KBr) Cm-1 : 3461 (Ar C-H), 3399 (NH), 3139 (NH2),
2219 (C C), 1890, 1657 (C=O), 1457, 1230, 1027, 911, 749, 687, 559cm-1. Anal.
C12H9N3O (M.W. 211.12). Calculated C 68.24, H 4.29, N 19.8. Found C 68.64, H 4.57, N
19.51.
4.6. In vitro anti-mycobacterial activity assay (M. bovis, M. tuberculosis, M.
avium)
20

M. bovis (BCG), M. tuberculosis (H37Ra) and M. avium (ATCC 25291) were
obtained from the American Type Culture Collection, Rockville, MD. The anti-
mycobacterial activity was determined using the microplate Alamar blue assay (MABA).
Test compounds were dissolved in DMSO at 100× the highest final concentration used, and
subsequent dilutions were performed in 7H9GC medium in 96-well plates. For these
experiments, each compound was tested at 100, 50, 10, and 1 μg/mL in triplicate. Frozen
mycobacterial inocula were diluted in medium 7H9GC and added to each well at a final
concentration of 2.5 × 105 CFU/mL. Control wells consisted of eight with bacteria alone (B)
and eight with media alone (M). Plates were incubated for 6 days, and then 20 μL of 10×
Alamar blue and 12.5 μL of 20% Tween-80 were added to one M and one B well. Wells
were observed for an additional 24-48 h for visual color change from blue to pink and read
by a spectrophotometer (at excitation 530 nm/525 nm and emission 590 nm/535 nm) to
determine OD values. If the B well became pink by 24 h (indicating growth), reagent was
added to the entire plate. If the B well remained blue, additional M and B wells were tested
daily until bacterial growth could be visualized by color change. After the addition of the
reagent to the plate, cultures were incubated for 24 h and plates were observed visually for
color change and also read by spectrophotometer. Visual MIC was defined as the lowest
concentration of a compound that prevented a color change from blue to pink. Percent
inhibition was calculated as (test well-M background/B well-M background) x 100. The
lowest drug concentration effecting an inhibition of ∼90% was considered as the MIC90.
Similar methodology was used for all (three) mycobacteria strains. Rifampicin and
clarithromycin were used as positive controls. As negative controls, DMSO was added to the
B well at a concentration similar to that of test wells; M wells served as negative controls. In
21

most of the experiments, the M wells gave an OD of 3000-4000 and the B wells had OD
values ranging between 60 000 and 100 000.
4.7. In vitro anti-tuberculosis activity of novel and standard compounds in
combination
For combination studies, we used the MABA assay employing similar
methodology as described above. The interaction of the compounds was determined by
calculating a synergy quotient using a formula reported earlier.19
4.8. In vitro cytotoxicity assay of test compounds
Cell viability was measured using the cell proliferation kit 1 (XTT;
Xenometric:Endotell), as per manufacturer’s instructions. Briefly, a 96-well plate was
seeded with Vero cells at a density of 2x105 cells per well. Cells were allowed to attach for
24 h, and the medium was replaced with medium containing compounds at concentrations of
200, 100, 50, 10 and 1 μg/mL. DMSO was also included as a control. Plates were incubated
for 3 days at 37° C then 10 μL XTT reagents were added per well and incubated for 4 h at 37
°C to obtain the color reaction. Plates were read on an ELISA plate reader (Abs 450-500
nm).
4.9. In vivo anti-mycobacterial activity
All animal experimental protocols used in this study were approved by the
University of Alberta Animal Care and Use Committee for Health Sciences, and conducted
in accordance with the guidelines of the University of Alberta, Edmonton, Canada, and the
Canadian Council on Animal Care. Five six-week old female BALB/c mice were purchased
from Charles River Laboratories and were allowed to acclimate for 1 week. Mice were
22

inoculated intravenously in the tail vein with 0.1 mL of bacterial suspension containing
approximately 0.5x 105 CFU of M. tuberculosis H37Ra in PBS. The infected mice were
divided into groups of 5 animals per group.
4.10. Administration of Compounds
The test compounds 3, 7 and control drug isoniazid were suspended in 0.5%
methyl-cellulose in saline. The drugs were frozen at -20° C and thawed each morning prior
to administration. Compounds 3 and 7 were administered orally at a dose of 25, 12.5 or 6.25
mg/kg; alone or in combination with isoniazid (1 mg/kg). Control animals received
equivalent volumes of diluents only. Treatment with compounds was initiated 4 days after
mycobacterial (H37Ra) challenge and continued until week 2 (total treatment days = 10).
Three days after the last treatment, mice were euthanized and lungs, liver, and spleen were
removed aseptically and individually homogenized. CFU counts per organ were determined
on 7H11 selective agar plates purchased from BD Biosciences. The plates were incubated at
37° C in ambient air for up to 4 weeks prior to counting the colonies.
4.11. Statistical Analyses
For animal experiments, standard randomization procedure was used in assigning
the groups to mice after infection. Statistical analysis was performed by 2way ANOVA
using Tukey’s multiple comparisons test using Graphpad Prism (Graphpad Software Inc).
‘*’, ‘**’, and ‘***’ indicate significant differences at P < 0.05, P < 0.01, and P < 0.001
respectively.
Acknowledgements
23

Part of this work was supported by the Canadian Institutes of Health Research
(CIHR) operating grant (MOP-49415) to RK.
24

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27

Graphical abstract
Investigation of 4-amino-5-alkynylpyrimidine-2(1H)-ones as antimycobacterial agents
Gaurav Garga, Milind Pandeb, Ambika Agrawalc, Jie Lid and Rakesh Kumar*c
2
3-7
28