The synthesis and evaluation of triazolopyrimidines as anti-tubercular agents

Article abstract of DOI:10.1016/j.bmc.2017.05.030

We identified a di-substituted triazolopyrimidine with anti-tubercular activity against Mycobacterium tuberculosis. Three segments of the scaffold were examined rationally to establish a structure-activity relationship with the goal of improving potency and maintaining good physicochemical properties. A number of compounds displayed sub-micromolar activity against Mycobacterium tuberculosis with no cytotoxicity against eukaryotic cells. Non-substituted aromatic rings at C5 and a two-carbon chain connecting a terminal aromatic at C7 were preferred features; the presence of NH at C7 and a lack of substituent at C2 were essential for potency. We identified compounds with acceptable metabolic stability in rodent and human liver microsomes. Our findings suggest that the easily-synthesized triazolopyrimidines are a promising class of potent anti-tubercular agents and warrant further investigation in our search for new drugs to fight tuberculosis.

Full text of DOI:10.1016/j.bmc.2017.05.030

Accepted Manuscript
The synthesis and evaluation of triazolopyrimidines as anti-tubercular agents
Edison S. Zuniga, Aaron Korkegian, Steven Mullen, Erik J. Hembre, Paul L.
Ornstein, Guillermo Cortez, Kallolmay Biswas, Naresh Kumar, Jeffrey Cramer,
Thierry Masquelin, Philip A. Hipskind, Joshua Odingo, Tanya Parish
PII:
S0968-0896(17)30266-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.030
BMC 13748
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
7 March 2017
10 May 2017
15 May 2017
Please cite this article as: Zuniga, E.S., Korkegian, A., Mullen, S., Hembre, E.J., Ornstein, P.L., Cortez, G., Biswas,
K., Kumar, N., Cramer, J., Masquelin, T., Hipskind, P.A., Odingo, J., Parish, T., The synthesis and evaluation of
triazolopyrimidines as anti-tubercular agents, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/
1
0.1016/j.bmc.2017.05.030
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The synthesis and evaluation of triazolopyrimidines as anti-tubercular agents
a
Authors: Edison S. Zuniga , Aaron Korkegian , Steven Mullen , Erik J. Hembre , Paul L. Ornstein ,
a
a
b
c
b
Guillermo Cortez , Kallolmay Biswas , Naresh Kumar , Jeffrey Cramer Thierry Masquelin , Philip A.
d
d
b
b
b
a
Hipskind , Joshua Odingo , and Tanya Parish
a*
a
TB Discovery Research, Infectious Disease Research Institute, 1616 Eastlake Avenue East, Seattle,
Washington 98102, USA
b
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA
c
Roosevelt University College of Pharmacy, Schaumburg, IL, 60173, USA
d
Jubilant Chemsys Limited, B-34, Sector 58, Noida, 201301, India
*Corresponding author
Professor Tanya Parish
E-mail: tanya.parish@idri.org

Abstract
We identified a di-substituted triazolopyrimidine with anti-tubercular activity against Mycobacterium
tuberculosis. Three segments of the scaffold were examined rationally to establish a structure-activity
relationship with the goal of improving potency and maintaining good physicochemical properties. A
number of compounds displayed sub-micromolar activity against Mycobacterium tuberculosis with no
cytotoxicity against eukaryotic cells. Non-substituted aromatic rings at C5 and a two-carbon chain
connecting a terminal aromatic at C7 were preferred features; the presence of NH at C7 and a lack of
substituent at C2 were essential for potency. We identified compounds with acceptable metabolic stability
in rodent and human liver microsomes. Our findings suggest that the easily-synthesized
triazolopyrimidines are a promising class of potent anti-tubercular agents and warrant further
investigation in our search for new drugs to fight tuberculosis.
Keywords: Tuberculosis, Mycobacterium tuberculosis, anti-tubercular activity, triazolopyrimidines
Graphical Abstract
O
para-Substituted phenylethylamines prefe
Longer chain homologs active

1. Introduction
Tuberculosis (TB) and its causative agent Mycobacterium tuberculosis present a serious threat to global
1
health. There are over 1 million deaths each year and approximately 9 million new cases . Taken together
with the estimate that a third of the world’s population is infected with M. tuberculosis and the existence
of drug-resistant strains of M. tuberculosis, it is apparent there is a pressing need for new therapies. To
address these needs, there has been an increased effort directed towards TB drug discovery in recent years
and a pipeline of new anti-TB drug candidates has started to emerge. The search for new molecular
scaffolds with potentially novel mechanisms of action remains a priority.
Triazolopyrimidines (TZPs) are a well-known scaffold in medicinal chemistry, and their utility is
exemplified by the discovery and development of novel agents to fight a wide range of diseases. For
2
example, TZPs possess anticancer activity, and have been used as phosphodiesterase inhibitors for
3
diabetes treatment. Recently, the first natural TZP, essramycin, was isolated and found to possess
4
antibacterial activity. A considerable effort has been made to develop TZPs with antimalarial activity.
5-7
Transition metal-containing TZPs have antiproliferative activity against Leishmania and Trypanosoma
8
cruzi, the protozoa that cause leishmaniasis and Chagas disease, respectively. In addition TZP
9
acylsulfonamides with anti-mycobacterial activity target acetohydroxyacid synthase. Similar compounds
10
were also identified in a phenotypic screening campaign against Mycobacterium.bovis BCG.
We identified a single TZP compound (1) from a whole-cell screen against M. tuberculosis which was
active in liquid culture (Fig. 1). The compound had good activity against M. tuberculosis with a
minimum inhibitory concentration (MIC) of 3.1 µM (Table 1). The compound was not cytotoxic, with an
IC50 of >100 µM against the HepG2 cell line; the selectivity index (SI), defined as the ratio of cytotoxicity

to MIC, was >32. Based on these data we initiated a structure-activity relationship (SAR) study around
this singleton.
Here, we present an exploratory study to understand the SAR of the TZP series. We identified key
functionalities and features necessary for anti-tubercular activity. In general, TZP compounds lack
cytotoxicity and display an encouraging metabolic stability profile. In addition, we demonstrated their
bactericidal activity against non-replicating bacteria.
Figure 1: Triazolopyrimidine (TZP) 1. Three segments are illustrated with boxes and numbered.
2. Results and Discussion
Our SAR investigation began with the design and synthesis of novel analogs based on modifications of
the core structure of compound 1. We set out to explore modifications of the core by way of heteroatom
replacement and the impact of chemical diversity at the C2, C5, and C7 positions.
2.1 Chemical synthesis
The general synthetic routes are shown in Schemes 1 and 2. We initially evaluated the SAR associated
with modifications at the C5 position of the TZP ring system. Synthesis of analogs 7-25 proceeded
through key 7-hydroxytriazolopyrimidine intermediates 4 (Scheme 1), which were obtained via the
9
condensation of the 1,3-di-keto compound 2 with the 5-amino-4H-1,2,4-triazole 3. Di-keto compounds
were prepared by treating the appropriate substituted ethanone with diethyl carbonate in the presence of
base. Chloro intermediates 5 were obtained upon treatment of 4 with phosphoryl chloride. Reaction of 5

with 4-methoxyphenethylamine gave C5 substituted triazolopyrimidines 7-25 in moderate to good yields.
Condensation between 3-(dimethylamino)-1-phenyl-propane-1-one and 3 directly gave 27.
Scheme 1: Synthesis of the triazolopyrimidine compounds. General conditions: (i) AcOH, 120 °C, 12-
3
16 h; (ii) POCl , 80 – 100 °C, 2 h; (iii) NMP, 80 -100 °C or room temperature.
Compound 5 served as a strategic intermediate from which a diverse set of C7 analogs could be prepared.
Condensation between 5 (R’ = phenyl) and a variety of amines (6) gave C7 substituted
triazolopyrimidines 27-48 with good yields. Similarly, reaction of 5 (R’ = 2-pyridinyl) with a variety of
amines gave C7 substituted analogs 51-60; the use of cyclic amines gave 62-64. Compound 28 was
obtained after treating 7-chloro-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (5a) with methanolic ammonia.
Synthesis of amide analogs 49 and 50 proceeded through 28 via EDCI-HOBt mediated coupling with the
appropriate substituted benzeneacetic acid. The alkylation of compound 1 using iodomethane afforded
analog 61.
As shown in Scheme 2, decoration of the C2 position of the TZP core proceeded through intermediate
66a-b, obtained from the condensation between ethyl 2-pyridylcarbonylacetate (2k) and a 2-substituted 5-
amino-1H-1,2,4-triazole (65a-b). Commercially available triazoles 65a and 65b were treated with 26 to
give 66a and 66b, respectively. Subsequent chlorination followed by treatment with 4-
methoxyphenylethylamine gave C2 substituted TZP analogs 67 and 68. Heterocyclic core replacement
analogues pyrazolopyrimidine 69 and imidazopyridine 70 were produced after manipulation of
commercially available intermediates 65c-d.

Scheme 2: Synthesis of triazolopyrimidine compounds exploring core modifications. General
conditions: (i) AcOH, 100 - 120 °C, 12 - 16 h; (ii) POCl , 80 - 100 °C, 2 h; (iii) amine 6, NMP, 80 - 100
C.
3
°
2.2 Structure-activity relationship (SAR) studies
We determined the activity of all the compounds synthesized against both M. tuberculosis and eukaryotic
cells (HepG2 cell line); we determined the MIC90 against M. tuberculosis, defined as the concentration
required to inhibit 90% growth in liquid medium, and the IC50 against HepG2 cells, defined as the
concentration required to reduce HepG2 viability by 50%. Selectivity index (SI) was calculated as
IC50/MIC90. We completed a systematic evaluation by modification of the TZP ring at the C5, C7, C2 and
core positions.
We first explored substitution of C5 phenyl with ortho- or para-electron-donating groups, which resulted
in a loss of activity (Table 1, compounds 7-10). The incorporation of fluorine on the para- or ortho-
positions (compounds 13 and 14) of the C5 phenyl ring was tolerated and maintained good separation
from cytotoxicity. This was not seen with chloride analogs 11 and 12 which had no activity. The strongly
3
deactivating para-CF phenyl moiety (compound 15) abrogated activity. Replacing the C5 phenyl with a
polar 2-pyridyl gave compound 16 with slightly increased potency while having no effect on cytotoxicity,
and provided a valuable point for further optimization. It is interesting to note that 3- or 4-pyridyl isomers
at C5 (17 and 18) resulted in loss of activity (Table 1).

Table 1: Examining Aromatic Moieties at C5
MIC
µM)
IC50
MIC
(µM)
IC50
Cpd
1
Ar
SI
Cpd
13
Ar
SI
(
(µM)
(µM)
3.1 ± 1.3
>100
>32
4.4 ± 2.0
89
>21
7
>20
>100
-
-
14
15
3.0 ± 0.21 >100
>30
-
8
9
>20
87
>20
>100
>20
>20
>20
37
-
-
-
16
17
18
0.83±0.26 >100
>130
1
1
0
1
>100
65
>20
>20
>100
69
-
-
1
2
>20
>100
-
19
>20
57
-
MIC90 is the minimum concentration required to inhibit growth of M. tuberculosis by 90%. MICs are the
average ± standard deviation of two independent experiments. IC50 is the concentration required to reduce
the viability of HepG2 cells by 50%. Selectivity index (SI) = IC50 /MIC90
.

No advantage was gained by replacing the aromatic group at C5 with either a linear alkyl (20, 21) or a
cyclohexyl group (25) and only small cyclic alkyl moieties such as the cyclopropyl (22), cyclopentyl 24
and cyclobutyl (23) analogs had good anti-tubercular activity (Table 2).
Table 2: Replacement of Aromatic Groups with Alkyl Moieties at C5
MIC
µM)
IC50
Cpd
1
R
SI
(
(µM)
3.1 ± 1.3
>100
>32
2
0
1
>20
>20
>100
>100
-
-
2
2
2
2
2
2
3
4
5
5.8 ± 1.6
>100
>17
>34
17
2.9 ± 0.71 >100
5.5 ± 0.07
>20
95
47
-
MIC90 is the minimum concentration required to inhibit growth of M. tuberculosis by 90%. MICs are the
average ± standard deviation of two independent experiments. IC50 is the concentration required to reduce
the viability of HepG2 cells by 50%. Selectivity index (SI) = IC50 /MIC90
.
We next explored modifications to the C7 position, while keeping a phenyl group at C5 (Table 3). Non-
aromatic moieties at C7 (e.g. compounds 27-35) did not show any anti-tubercular activity, except for the
ethyl tethered cyclohexyl analog (36).

Table 3: Alkyl Chains at C7
MIC IC50
MIC
(µM)
IC50
Cpd
R
SI
-
Cpd
R
SI
-
(µM) (µM)
(µM)
2
2
7
8
>20
>20
32
33
>20
98
>20
>20
-
>20
>100
-
2
3
3
9
0
1
>20
>20
>20
>20
37
-
-
-
34
35
36
>20
>20
>100
57
-
-
94
17 ± 2.1
>100
>6
MIC90 is the minimum concentration required to inhibit growth of M. tuberculosis by 90%. MICs are the
average ± standard deviation of two independent experiments. IC50 is the concentration required to reduce
the viability of HepG2 cells by 50%. Selectivity index (SI) = IC50 /MIC90
.
We next evaluated the effect of chain length at C7 (Table 4). Compounds 37 (with no tether), 38 (with a
one carbon tether), and 40 (with a three carbon tether) were all inactive. A reduction in potency was
observed for the unsubstituted terminal phenyl group as in compound 39 compared to the para-methoxy
analog 1. This pointed to the importance of para-substitution for activity and selectivity. Polar aromatics
at the terminal end of the C7 alkyl chain were not tolerated as demonstrated by the inactive pyridyl
analogs 41-43. Para-substituted analogues 44-48 were prepared, and the overall potency was rescued in
all cases except for the para-fluoro analog 46. C7 amide analogs (49 and 50) retained activity and were
not cytotoxic indicating that properties of the aniline nitrogen at C7 can be modified with little penalty to
activity.

Table 4: C7 Linker Modifications with C5 Phenyl
MIC
IC50
MIC
(µM)
IC50
Cpd
R
SI
Cpd
44
R
SI
(µM) (µM)
(µM)
37
38
39
40
41
42
43
>20
>20
49
43
-
1.4 ± 0.5
0.74±0.1
>20
53
43
-
3.5
-
45
46
47
48
49
50
>100
50
>138
-
7.7±0.7
>20
27
8.7
2.7 ± 0.7
3.9 ± 1.1
1.5 ± 0.7
10 ± 1.1
>100
>100
>100
>100
>38
>27
>73
>10
>20
>100
>100
>100
-
>20
-
>20
-
MIC90 is the minimum concentration required to inhibit growth of M. tuberculosis by 90%. MICs are the
average of two independent experiments. IC50 is the concentration required to reduce the viability of
HepG2 cells by 50%. Selectivity index (SI) = IC50 /MIC90
.
Thus far we demonstrated that optimal anti-tubercular activity was found in the 2-pyridyl analogue 16.
As with the C5 phenyl derivatives, we examined modifications to the terminal aromatic ring of the C7
side chain while keeping the 2-pyridyl at C5 constant (Table 5). A variety of para-substituted aromatic
groups were tolerated at the terminal end of the C7 side chain resulting in analogs (55-60) with
comparable or improved potency and good separation from cytotoxicity. It is worthy to note that analogs

containing terminal pyridyl moieties on the C7 side chain (52-54) lost activity, as observed in the C5
phenyl series, demonstrating that polar residues are not tolerated in this region for either series.
Table 5: C7 Modifications with 2-Pyridyl at C5
MIC
µM)
IC50
MIC
(µM)
IC50
Cpd
R
SI Cpd
R
SI
(
(µM)
(µM)
51
52
53
54
2.4 ± 1.3
>100
>100
>100
>100
>46
56
57
58
59
0.2 ± 0.02
4.8 ± 1.3
0.98 ±0.4
1.6 ± 1.1
44
220
>20
-
-
-
81
17
>20
>100
>100
>119
>72
>20
Cl
5
5
0.52± 0.1 >100 >196
60
0.76 ±0.25 >100
>135
MIC90 is the minimum concentration required to inhibit growth of M. tuberculosis by 90%. MICs are the
average ± standard deviation of two independent experiments. IC50 is the concentration required to reduce
the viability of HepG2 cells by 50%. Selectivity index (SI) = IC50 /MIC90
.
We investigated other SAR elements of the spacer at C7 (Table 6). The N-methylated analog 61 had a
loss of activity, an indication that H-bonding may be important for the TZP compounds binding to their
target (this could also be due to unfavorable steric interactions). Introducing rigidity on the amine tether
as in pyrrolidine 62, piperidine 63 and morpholino 64 was not tolerated. It has been previously reported
that substitution on the C2 position of the TZP scaffold gave compounds with potent antimalarial activity
7
and with good metabolic stability. Based on these findings and with the goal of identifying active and
metabolically stable compounds, we prepared and tested the 2-methyl (67) and 2-phenyl (68) analogs.
Analog 67 had comparable activity to our original hit, but analog 68 showed an MIC >20 µM.

1
1
12, 13
Pyrazolopyrimidine and imidazopyridine
compounds have also been reported to possess potent anti-
tubercular activity. Analog 69 with a pyrazolopyrimidine core demonstrated comparable activity to
original hit 1, but with increased cytotoxicity, whereas the imidazopyrimidine-based analog 70 had
excellent anti-tubercular activity as well as good separation from cytotoxicity.
Table 6: Constraining C7 Side Chain and Core Modification
MIC IC50
MIC
(µM)
IC50
Cpd
Structure
SI Cpd
Structure
SI
(µM) (µM)
(µM)
6
1
>20
>20
17
23
-
67
3.9 ± 0.21 >100
>26
6
2
-
68
>20
>100
-
6
3
>20
16
-
69
2.5 ± 0.15
23
9
6
4
>20
50
-
70
0.22±0.07
30
142
MIC90 is the minimum concentration required to inhibit growth of M. tuberculosis by 90%. MICs are the
average ± standard deviation of two independent experiments. IC50 is the concentration required to reduce
the viability of HepG2 cells by 50%. Selectivity index (SI) = IC50 /MIC90
.

2.3 Microsomal stability and in vivo pharmacokinetic (PK) studies
Based on in silico ADME predictions, three compounds were chosen to cover a range of cLogP values
and were evaluated for their in vitro microsomal stability (Table 7). Rapid metabolism of compound 44
3
was observed, in rodent and human liver microsomes. The para-OCF analog 48 was also rapidly
metabolized. The amide (49) had improved in vitro microsomal stability, with only 12% loss after 30 min
in mouse microsomes, 22% loss in rat microsomes and 31% loss in human microsomes. The difference in
stability for these three compounds is probably due to presence of more than one oxidatively-labile
carbon in 44 and 48 (both have a two carbon linker), while 49 has only one such soft spot, although these
have not been confirmed with metabolite identification studies.
The oral exposure of the three compounds was evaluated in male mice by comparing exposures following
oral (PO) and intravenous (IV) administration of 10 and 1 mg/kg doses of compounds, respectively (Table
7). Overall, the three compounds showed poor to moderate in vivo mouse PK properties. Compound 44
had an AUC = 676 nM*h, and rapid IV clearance (182 mL/min/kg) as predicted by mouse microsomes.
Compound 48 had more promising mouse PK with greater oral exposure (AUC 2750 nM*h), a longer
half-life, and slower IV clearance (33.5 mL/min/kg). Surprisingly, despite showing good in vitro
microsomal metabolic stability, amide 49 had the lowest oral exposure and fastest IV clearance rates in
vivo compared to the two other compounds.
Table 7: In vitro ADME and in vivo PK
PO
AUC
nM*h)
PO
Cmax Tmax t1/2 (mL/min/kg)
PO
PO IV clearance
% turnover by liver
microsomes in 30 min
(
(nM)
(h)
(h)
mouse
rat
human mouse Predicted
a b
c
Cpd
Fu,pl
0.015
0.007
Fu,pl
0.020
0.006
clogP
4.33
5.25
4
4
4
8
98.2
52.1
99.2
52.7
68.1
32.8
676
2750
604
958
0.25
0.75
1.05
2.49
182
33.5

49
12.5
21.8
30.6
ND
0.048
3.25
187
62.8
1.5
ND
248
a
b
Fu,pl is the fraction unbound in plasma. Predicted value using a QSAR model built based on data
c
generated for >3000 compounds measured internally (unpublished). clogP was predicted by Chemaxon
model (www.chemaxon.com).
2.4 Activity Spectrum
Three compounds (16, 48 and 49) were selected for testing against other organisms based on good
activity in liquid and solid medium (MIC99 < 20 µM) against M. tuberculosis (Table 8). We tested against
a non-pathogenic mycobacterial species (Mycobacterium smegmatis), Escherichia coli (Gram negative),
Pseudomonas aeruginosa (Gram negative), Bacillus subtilis (Gram positive) and yeast (Saccharomyces
cerevisiae) (Table 8). All three compounds had activity against M. tuberculosis, but no activity against
any of the other species. Thus, the TZP compounds are selective for M. tuberculosis.
Table 8: Spectrum of activity
MIC99 (µM)
Cpd
M. tuberculosis
M. smegmatis
E. coli
P. aeruginosa
S. cerevisiae
B. subtilis
1
4
4
6
8
9
13
>100
>100
>100
>100
>100
1.6
3.1
>10
>10
>10
>10
>100
>10
>10
>10
>10
>10
MIC99 is the lowest concentration of compound, which yielded less than 1% growth.
2.5 Activity against replicating and non-replicating M. tuberculosis
We also determined the effectiveness of the three compounds (16, 48 and 49) in killing replicating and
non-replicating M. tuberculosis. Compounds exhibited static activity against replicating bacteria,
preventing growth, but no kill was noted over 21 days. In contrast, we did note killing against non-
replicating bacteria (starvation conditions) for all three compounds, with a 2-3 log kill over 14-21 days.

3.0 Conclusion
We conducted a systematic exploration of the triazolopyrimidine scaffold for activity against M.
tuberculosis. Overall, the compounds in this series show good activity and selectivity. Our initial
explorations suggest that non-substituted aromatic rings at C5, and a two-carbon chain connecting a
terminal aromatic at C7 are preferred features for potency against M. tuberculosis and separation from
cytotoxicity. The presence of NH at C7 and a lack of substituent at C2 are essential for potency of the

molecule. Heteroatom replacement or homologation of the scaffold is well tolerated in the series. We
have identified compounds with improved metabolic stability in rodent and human liver microsomes,
however, oral exposure and clearance remains an issue for this series. We feel that these issues can be
improved through further SAR exploration. Thus, there is substantial promise in developing the TZP
series that warrants further investigation as novel tools in our drug arsenal to combat tuberculosis.
4. 0 Materials and Methods
4.1 Determination of minimum inhibitory concentration (MIC)
1
4
MICs were determined against M. tuberculosis H37Rv as described in . Briefly, M. tuberculosis was
grown in Middlebrook 7H9 medium containing 10% OADC (oleic acid, albumin, dextrose, catalase)
supplement (Becton Dickinson) and 0.05% w/v Tween 80 (7H9-Tw-OADC) under aerobic conditions.
Bacterial growth was measured by OD590 after 5 days of incubation at 37 °C. Curves were fitted using the
Levenberg–Marquardt algorithm. MIC90 was defined as the minimum concentration required to inhibit
growth of M. tuberculosis by 90%.
4.2 Determination of cytotoxicity (IC50)
Cytotoxicity was assessed using the HepG2 cell line under replicating conditions. HepG2 cells were
grown in DMEM, High Glucose, GlutaMAX™ (Invitrogen), 10% fetal bovine serum (FBS), and 1X
penicillin-streptomycin solution (100 U/mL). Cells were incubated with compounds for 2 days at 37 °C
(final DMSO concentration of 1%), 5% CO2. CellTiter-Glo® Reagent (Promega) was added and relative
luminescent units (RLU) measured to assess cell viaility. Inhibition curves were fitted using the
Levenberg–Marquardt algorithm. IC50 is the concentration required to reduce cell viability after 2 days by
50%. Controls were 1% DMSO only (growth control) and staurosporine (positive control).
4.3 Determination of minimum inhibitory concentration on solid medium (MIC)

The serial proportion method was used to determine MIC99 on solid medium for a variety of
15
2
representative species . M. tuberculosis H37Rv and Mycobacterium smegmatis mc 155 were grown in
Middlebrook 7H9 medium + 10% v/v OADC (oleic acid, albumin, dextrose, catalase) supplement
(Becton Dickinson) and on Middlebrook 7H10 medium + 10% v/v OADC. Plates were incubated at 37°C
for 4 weeks and 3-4 days respectively; Escherichia coli DH5α and Staphylococcus aureus RN4220 were
grown on LB agar and incubated at 37°C for 1 and 2 days respectively; Pseudomonas aeruginosa
HER1018 (PAO1) was grown on tryptic soy agar and incubated at 37°C for 1 day; Bacillus subtilis
Marburg was grown in on nutrient agar and incubated at 28°C for 3-4 days; Saccharomyces cerevisiae
Y187 was grown on YPD agar plus 0.003% w/v adenine hemi-sulfate and incubated at 30°C for 3-4 days.
The MIC99 was the lowest concentration of compound, which yielded less than 1% growth relative to no-
compound control.
4.4 Kill kinetics
To determine kill kinetics, replicating bacteria, a late log phase culture (OD590 0.6-1.0) of M. tuberculosis
was adjusted to an OD590 of 0.1 in 7H9-Tw-OADC; 50 µL was used to inoculate 5 mL 7H9-Tw-OADC
containing compounds. For non-replicating conditions, bacteria were incubated at 37 °C for 14 days in
PBS + 0.05% w/v Tyloxapol (PBS-Ty) at an OD590 of 0.1 and then compounds were added (final DMSO
concentration of 2%). Cultures were incubated standing at 37 °C. Serial dilutions were plated on 7H10-
OADC agar to determine CFU/mL.
4.5 Compound synthesis
4.5.1 General Methods
1
13
3 6
H and C NMR spectral data were recorded in CDCl or DMSO-d on a 300 or 400 MHz Bruker NMR
spectrometer. Column chromatography was conducted on silica gel (100–300 mesh). Reactions were
monitored using thin-layer chromatography (TLC) on silica gel plates. HPLC analysis was conducted on
an Agilent 1100 series LC system (Agilent ChemStation Rev.A.10.02; Phenomenex-Luna-C18, 4.8 mm ×
2
150 mm, 5 µm, 1.0 mL/min, UV 254nm, room temperature) with MeCN/H O (0.05% TFA or HCOOH

buffer) gradient elution. HPLCMSwas performed on a Gilson 321 HPLC with detection performed by a
Gilson 170 DAD and a Finnigan AQA mass spectrometer operating in electrospray ionisation mode using
a Phenomenex Gemini C18 150x4.6mm column. Purity was determined using a Waters Acquity UPLC
system equipped with a BEH C18 1.7µm 2.1 x 100 mm column.
4
.5.2 General procedure for the synthesis of N-phenethyl-5-phenyl-[1,2,4]triazolo[1,5-
a]pyrimidin-7-amines (1, 27-50).
-Chloro-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine 4 (1.0 eq) was was taken in NMP in a 100 mL round
7
2
bottom flask under N . To it was added amine precursor 6 (1.2 eq). The reaction mixture was heated at
o
0-120 C or rt until complete by TLC analysis. The reaction mixture was then added to ice-cooled water
9
2 4
and extracted with EtOAc. The organic layer was dried over anhydrous Na SO and concentrated under
reduced pressure. The crude was purified by flash chromatography on silica gel (100-200 mesh) using
EtOAc-hexane or MeOH-DCM as eluent to afford compounds 1, 27-50.
4
.5.3 General procedure for the synthesis of N-substituted-5-(pyridin-2-yl)-
1,2,4]triazolo[1,5-a]pyrimidin-7-amine (16, 51-60).
-Chloro-5-(pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidine 5k (1.0 eq) was was taken in NMP in a 100
[
7
2
mL round bottom flask under N . To it was added amine precursor (1.2 eq). The reaction mixture was
o
heated at 90-100 C or rt until complete by TLC analysis. The reaction mixture was then added to ice-
cooled water and extracted with EtOAc. The organic layer was dried over anhydrous Na
2 4
SO and
concentrated under reduced pressure. The crude was purified by flash chromatography on silica gel (100-
200 mesh) using EtOAc-hexane or MeOH-DCM as eluent to afford compounds 16, 51-60.
4.5.4 N-(4-methoxyphenethyl)-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (1): 7-chloro-5-
phenyl-[1,2,4]triazolo[1,5-a]pyrimidine 5 (500 mg, 2.2 mmol) was taken in NMP (5 mL) in a 50 mL
round bottom flask under N . To it was added 2-(4-methoxyphenyl)ethan-1-amine (365 mg, 2.4 mmol).
2
o
The reaction mixture was heated at 100 C and monitored by TLC analysis (EtOAc, 100%) until

completion. The reaction mixture was then poured into ice water (50 g) and extracted with EtOAc (2 x 50
mL). The combined organic layer was dried over anhydrous Na SO and concentrated under reduced
pressure. The crude was purified by flash chromatography on silica gel (100-200 mesh) using 30%
2
4
o
1
EtOAc-hexane as eluent to afford 1 as a white solid (300 mg, 40%). M.P. 163-164 C. H NMR (400
MHz, DMSO-d ): δ 8.46 (s, 1H), 8.34 (bs, 1H), 8.15-8.17 (m, 2H), 7.52-7.53 (m, 3H), 7.23 (d, J = 8.3 Hz,
H), 6.84 (d, J = 7.2 Hz, 3H), 3.73-3.76 (m, 2H), 3.66 (s, 3H), 2.92-2.96 (m, 2H); C NMR (100 MHz,
DMSO-d ): δ 160.2, 157.8, 155.5, 154.7, 147.9, 137.6, 130.7, 130.2, 129.9, 128.5, 127.4, 113.7, 84.8,
4.9, 43.2, 33.7. LCMS (ESI) m/z 346.45.
6
1
3
2
6
5
4.5.5 Ethyl 3-(2-methoxyphenyl)-3-oxopropanoate (2c). Sodium hydride (480 mg, 20.0 mmmol) was
o
and cooled it down to 0 C. To it was
taken in dry THF (18 mL) in a 100 mL round bottom flask under N
2
added a solution of ethyl 3-(2-methoxyphenyl)-3-oxopropanoate (1.0 g, 6.7 mmol) in THF (2 mL). The
reaction mixture was stirred at rt for 30 min followed by the addition of diethyl carbonate (3.2 mL, 26.8
mmol). The reaction mixture was then stirred at rt for 14 h. Ice-cooled water was added dropwise to
quench the reaction. It was extracted with EtOAc (3 x 75 mL). The combined organic layer was dried
2 4
over anhydrous Na SO and concentrated under reduced pressure. The crude was further purified by flash
chromatography on silica gel (60-120 mesh) using 10% hexane-EtOAc as eluent to afford 2c as a
+
colourless liquid (1.3 g, 88%). LC-MS (ESI) m/z 223.32 [M+H ]; 97% (purity).
4.5.6 Ethyl 3-oxo-3-(p-tolyl)propanoate (2d). Sodium hydride (2.68 g, 112 mmmol) was taken in dry
o
and cooled it down to 0 C. To it was added a
DMF (25 mL) in a 250 mL round bottom flask under N
2
solution of 1-(p-tolyl)ethan-1-one (5.0 g, 37.3 mmol) in DMF (5 mL). The reaction mixture was stirred at
rt for 30 min followed by the addition of diethyl carbonate (18 mL, 149.2 mmol). The reaction mixture
was then stirred at rt for 16h. Ice-cooled water was added dropwise to quench the reaction. It was
extracted with EtOAc (3 x 100 mL). The combined organic layer was dried over anhydrous Na
2 4
SO and
concentrated under reduced pressure. The crude was further purified by flash chromatography on silica

gel (60-120 mesh) using 10% hexane-EtOAc as eluent to afford 2d as a colourless liquid (6.2 g, 81%).
+
LCMS (ESI) m/z 207.32 [M+H ]; 84.88% (purity).
4.5.7 Ethyl 3-(4-ethylphenyl)-3-oxopropanoate (2e). Sodium hydride (161 mg, 6.74 mmmol) was taken
o
and cooled it down to 0 C. To it was added
in dry THF (8 mL) in a 100 mL round bottom flask under N
2
a solution of 1-(4-ethylphenyl)ethan-1-one (500 mg, 3.37 mmol) in THF (2 mL). The reaction mixture
was stirred at rt for 30 min followed by the addition of diethyl carbonate (1.6 mL, 13.5 mmol). The
reaction mixture was then stirred at rt for 12 h. Ice-cooled water was added dropwise to quench the
reaction. It was extracted with EtOAc (3 x 50 mL). The combined organic layer was dried over anhydrous
Na
2
SO
4
2
and concentrated under reduced pressure. The crude was triturated with Et O afford 2e as a
+
brown solid (500 mg, 67%). LCMS(ESI) m/z 221.29 [M+H ]; 50% (purity).
4.5.8 Ethyl 3-(2-fluorophenyl)-3-oxopropanoate (2i). Sodium hydride (1.74 g, 72.4 mmmol) was taken
o
and cooled it down to 0 C. To it was
in dry THF (30 mL) in a 100 mL round bottom flask under N
2
added a solution of 1-(2-fluorophenyl)ethan-1-one (5.0 g, 36.2 mmol) in THF (5 mL). The reaction
mixture was stirred at rt for 30 min followed by the addition of diethyl carbonate (17.5 mL, 144.8 mmol).
The reaction mixture was then stirred at rt for 12h. Ice-cooled water was added dropwise to quench the
reaction. It was extracted with EtOAc (3 x 75 mL). The combined organic layer was dried over anhydrous
Na
2
SO
4
2
and concentrated under reduced pressure. The crude was triturated with Et O afford 2i as a brown
+
liquid (4.0 g, 52%). MS (ESI) m/z 211.17 [M+H ].
4.5.9 Ethyl 3-oxo-3-(4-(trifluoromethyl)phenyl)propanoate (2j). Sodium hydride (2.55 g, 106.4
2
mmmol) was taken in dry THF (40 mL) in a 250 mL round bottom flask under N and cooled it down to 0
o
C. To it was added a solution of 1-(4-(trifluoromethyl)phenyl)ethan-1-one (10 g, 53.2 mmol) in THF (10
mL). The reaction mixture was stirred at rt for 30 min followed by the addition of diethyl carbonate (25.8
mL, 212.8 mmol). The reaction mixture was then stirred at rt for 14 h. Ice-cooled water was added
dropwise to quench the reaction. It was extracted with EtOAc (3 x 50 mL). The combined organic layer
was dried over anhydrous Na
Et
2
SO
4
and concentrated under reduced pressure. The crude was triturated with
+
O afford 2j as a brown solid (10.0 g, 72%). LCMS(ESI) m/z 259.14 [M-H ]; 61% (purity).
2

4.5.10 Ethyl 3-oxo-3-(pyridin-3-yl)propanoate (2l). Sodium hydride (2.9 g, 124 mmmol) was taken in
o
and cooled it down to 0 C. To it was added a
dry THF (45 mL) in a 250 mL round bottom flask under N
2
solution of 1-(pyridin-3-yl)ethan-1-one (5.0 g, 41.2 mmol) in THF (5 mL). The reaction mixture was
stirred at rt for 30 min followed by the addition of diethyl carbonate (20 mL, 165.0 mmol). The reaction
mixture was then stirred at rt for 14 h. Ice-cooled water was added dropwise to quench the reaction. It was
2 4
extracted with EtOAc (3 x 100 mL). The combined organic layer was dried over anhydrous Na SO and
concentrated under reduced pressure. The crude was further purified by flash chromatography on silica
gel (60-120 mesh) using 10% hexane-EtOAc as eluent to afford 2l as a colorless liquid (4.1 g, 52%).
+
LCMS(ESI) m/z 194.25 [M+H ]; 88% (purity).
4.5.11 Ethyl 3-cyclobutyl-3-oxopropanoate (2r). Sodium hydride (2.44 g, 101.9 mmmol) was taken in
o
and cooled it down to 0 C. To it was added a
dry THF (55 mL) in a 250 mL round bottom flask under N
2
solution of 1-cyclobutylethan-1-one (5.0 g, 50.9 mmol) in THF (5 mL). The reaction mixture was stirred
at rt for 30 min followed by the addition of diethyl carbonate (24.7 mL, 203.6 mmol). The reaction
mixture was then stirred at rt for 14 h. Ice-cooled water was added dropwise to quench the reaction. It was
2 4
extracted with EtOAc (3 x 200 mL). The combined organic layer was dried over anhydrous Na SO and
concentrated under reduced pressure. The crude was further purified by flash chromatography on silica
gel (60-120 mesh) using 10% hexane-EtOAc as eluent to afford 2r as a colorless semi-solid (2.6 g, 30%).
+
LCMS(ESI) m/z 169.07 [M-H ]; 80% (purity).
4
2
.5.12 Synthesis of 5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4): Ethyl 3-oxo-3-phenylpropanoate
(10.0 g, 52.1 mmol) was taken in AcOH (50 mL) in a 250 mL round bottom flask under N . To it was
2
o
added 1H-1,2,4-triazol-5-amine 3 (0.4 mL, 62.5 mmol). The reaction mixture was heated at 120 C for
14h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent and
triturated with diethyl ether. This was finally dried under high vaccum which affored a white solid (10.5
g, 95%). This was then used in the next step without any further purification. LCMS(ESI) m/z 213.09
+
M+H ]; 59.54% (purity).
[

4
5
1
.5.13 5-Phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4a). Ethyl 3-oxo-3-phenylpropanoate 2 (10.0 g,
2.1 mmol) was taken in AcOH (50 mL) in a 250 mL round bottom flask under N . To it was added 1H-
2
o
,2,4-triazol-5-amine (0.4 mL, 62.5 mmol). The reaction mixture was heated at 120 C for 14 h. The
reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent and triturated with
diethyl ether. This was finally dried under high vacuum which afforded a white solid (10.5 g, 95%). This
+
was then used in the next step without any further purification. LCMS(ESI) m/z 213.09 [M+H ]; 59.54%
(purity).
4.5.14 5-(4-Methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4b). Ethyl 3-(4-methoxyphenyl)-3-
2
oxopropanoate (1.0 g, 4.5 mmol) was taken in AcOH (5 mL) in a 50 mL round bottom flask under N . To
o
it was added 1H-1,2,4-triazol-5-amine (416 mg, 4.9 mmol). The reaction mixture was heated at 120 C for
12 h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent and
triturated with diethyl ether. This was finally dried under high vacuum which afforded 4b as a brown
solid (510 mg, 47%). This was then used in the next step without any further purification. LCMS(ESI)
+
m/z 243.10 [M+H] ; 80.54% (purity).
4.5.15 5-(2-Methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4c). Ethyl 3-(2-methoxyphenyl)-3-
oxopropanoate 2c (1.5 g, 6.7 mmol) was taken in AcOH (15 mL) in a 100 mL round bottom flask under
2
N . To it was added 1H-1,2,4-triazol-5-amine 2 (681 mg, 8.1 mmol). The reaction mixture was heated at
o
20 C for 14 h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic
1
solvent and triturated with diethyl ether. This was finally dried under high vacuum which afforded 4c as
an off-white solid (1.2 g, crude). This was then used in the next step without any further purification.
+
LCMS(ESI) m/z 243.11 [M+H ]; 95.10% (purity).
4.5.16 5-(p-Tolyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4d). Ethyl 3-oxo-3-(p-tolyl)propanoate 2d (6.0
g, 29.1 mmol) was taken in AcOH (40 mL) in a 250 mL round bottom flask under N . To it was added
2
o
H-1,2,4-triazol-5-amine (2.9 g, 34.9 mmol). The reaction mixture was heated at 120 C for 14 h. The
1
reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent and triturated with
diethyl ether. This was finally dried under high vacuum which afforded 4d as a white solid (1.8 g, 27%).

+
This was then used in the next step without any further purification. LCMS(ESI) m/z 227.22 [M+H ];
75.24% (purity).
4.5.17 5-(4-Ethylphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4e). Ethyl 3-(4-ethylphenyl)-3-
oxopropanoate 2e (500 mg, 2.27 mmol) was taken in AcOH (6 mL) in a 50 mL round bottom flask under
2
N . To it was added 1H-1,2,4-triazol-5-amine (286 mg, 3.4 mmol). The reaction mixture was heated at
o
15 C for 16 h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic
1
solvent and triturated with diethyl ether. This was finally dried under high vacuum which afforded 4e as a
brown solid (400 mg, 73%). This was then used in the next step without any further purification.
+
LCMS(ESI) m/z 239.20 [M-H ]; 45% (purity).
4.5.18 5-(4-Chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4f). Ethyl 3-(4-chlorophenyl)-3-
2
oxopropanoate (1.0 g, 4.4 mmol) was taken in AcOH (5 mL) in a 50 mL round bottom flask under N . To
o
it was added 1H-1,2,4-triazol-5-amine (406 mg, 4.8 mmol). The reaction mixture was heated at 120 C for
16 h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent and
triturated with diethyl ether. This was finally dried under high vacuum which afforded 4f as a white solid
(560 mg, 51%). This was then used in the next step without any further purification. LCMS(ESI) m/z
+
47.15 [M+H ]; 98.87% (purity).
2
4.5.19 5-(4-Fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4h). Ethyl 3-(4-fluorophenyl)-3-
2
oxopropanoate (2.0 g, 9.5 mmol) was taken in AcOH (10 mL) in a 50 mL round bottom flask under N .
To it was added 1H-1,2,4-triazol-5-amine 2 (958 mg, 11.4 mmol). The reaction mixture was heated at 120
o
C for 12 h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent
and triturated with diethyl ether. This was finally dried under high vacuum which afforded 4h as a yellow
solid (1.1 g, 50%). This was then used in the next step without any further purification. LCMS(ESI) m/z
+
31.25 [M+H ]; 79.73% (purity).
2
4.5.20 5-(2-Fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4i). Ethyl 3-(2-fluorophenyl)-3-
oxopropanoate 2i (4.0 g, 19.0 mmol) was taken in AcOH (20 mL) in a 100 mL round bottom flask under
. To it was added 1H-1,2,4-triazol-5-amine (1.92 g, 22.8 mmol). The reaction mixture was heated at
N
2

o
15 C for 12 h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic
1
solvent and triturated with diethyl ether. This was finally dried under high vacuum which afforded 4i as a
yellow solid (800 mg, 18%). This was then used in the next step without any further purification.
+
LCMS(ESI) m/z 231.06 [M+H ]; 54% (purity).
4.5.21 5-(4-(Trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4j). Ethyl 3-oxo-3-(4-
(trifluoromethyl)phenyl)propanoate 2j (10.0 g, 38.5 mmol) was taken in AcOH (50 mL) in a 250 mL
2
round bottom flask under N . To it was added 1H-1,2,4-triazol-5-amine (4.85 g, 57.7 mmol). The reaction
o
mixture was heated at 115 C for 16 h. The reaction mixture was then evaporated to dryness using toluene
as an azeotropic solvent and triturated with diethyl ether. This was finally dried under high vacuum which
afforded 4j as a yellow solid (6.0 g, 56%). This was then used in the next step without any further
+
purification. LCMS(ESI) m/z 279.43 [M-H ]; 78% (purity).
4.5.22
5-(Pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol
(4k).
Ethyl
3-oxo-3-(pyridin-2-
yl)propanoate 2k (8.0 g, 41.4 mmol) was taken in AcOH (50 mL) in a 250 mL round bottom flask under
2
N . To it was added 1H-1,2,4-triazol-5-amine (4.2 g, 49.7 mmol). The reaction mixture was heated at 120
o
C for 16 h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent
and triturated with diethyl ether. This was finally dried under high vacuum which afforded 4k as a deep
brown solid (4.5 g, 51%). This was then used in the next step without any further purification. MS (ESI)
+
m/z 212.2 [M-H ].
4.5.23
5-(Pyridin-3-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol
(4l).
Ethyl
3-oxo-3-(pyridin-3-
yl)propanoate 2l (4.0 g, 20.7 mmol) was taken in AcOH (40 mL) in a 250 mL round bottom flask under
2
N . To it was added 1H-1,2,4-triazol-5-amine (2.08 g, 24.8 mmol). The reaction mixture was heated at
o
20 C for 14 h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic
1
solvent and triturated with diethyl ether. This was finally dried under high vacuum which afforded 4l as a
white solid (2.1 g, 48%). This was then used in the next step without any further purification. LCMS(ESI)
+
m/z 214.13 [M+H ]; 62% (purity).

4.5.24 5-(Pyridin-4-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4m). Ethyl 3-oxo-3-(pyridin-4-
2
yl)propanoate (2.0 g, 10.4 mmol) was taken in AcOH (10 mL) in a 50 mL round bottom flask under N .
o
To it was added 1H-1,2,4-triazol-5-amine (1.04 g, 12.4 mmol). The reaction mixture was heated at 110 C
for 16 h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent and
triturated with diethyl ether. This was finally dried under high vacuum which afforded 4m as a yellow
solid (1.2 g, 54%). This was then used in the next step without any further purification. LCMS(ESI) m/z
+
14.19 [M+H ]; 56% (purity).
2
4.5.25 5-Benzyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4n). Ethyl 3-oxo-4-phenylbutanoate (1.5 g, 7.2
mmol) was taken in AcOH (8 mL) in a 50 mL round bottom flask under N . To it was added 1H-1,2,4-
2
o
triazol-5-amine (733 mg, 8.7 mmol). The reaction mixture was heated at 115 C for 16 h. The reaction
mixture was then evaporated to dryness using toluene as an azeotropic solvent and triturated with diethyl
ether. This was finally dried under high vacuum which afforded 4n as a white solid (910 mg, 55%). This
was then used in the next step without any further purification.
4.5.26 5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4o). Ethyl 3-oxobutanoate (1.0 g, 7.7 mmol) was
taken in AcOH (5 mL) in a 50 mL round bottom flask under N . To it was added 1H-1,2,4-triazol-5-
2
o
amine (646 mg, 7.7 mmol). The reaction mixture was heated at 120 C for 16 h. The reaction mixture was
then evaporated to dryness using toluene as an azeotropic solvent and triturated with diethyl ether. This
was finally dried under high vacuum which afforded 4o as a white solid (610 mg, 53%). This was then
+
used in the next step without any further purification. LCMS (ESI) m/z 151.06 [M+H ]; 78% (purity).
4.5.27 5-Ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4p). Ethyl 3-oxopentanoate (200 mg, 1.4 mmol)
was taken in AcOH (1.0 mL) in a 25 mL round bottom flask under N . To it was added 1H-1,2,4-triazol-
2
o
-amine (139 mg, 1.6 mmol). The reaction mixture was heated at 120 C for 16 h. The reaction mixture
5
was then evaporated to dryness using toluene as an azeotropic solvent and triturated with diethyl ether.
This was finally dried under high vacuum which afforded 4p as an off-white solid (110 mg, 48%). This
+
was then used in the next step without any further purification. LCMS(ESI) m/z 165.43 [M+H ]; 90%
(purity).

4.5.28 5-Cyclobutyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4r). Ethyl 3-cyclobutyl-3-oxopropanoate 2r
(5.0 g, 29.4 mmol) was taken in AcOH (25 mL) in a 100 mL round bottom flask under N . To it was
2
o
added 1H-1,2,4-triazol-5-amine (2.9 g, 35.2 mmol). The reaction mixture was heated at 110 C for 14 h.
The reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent and triturated
with diethyl ether. This was finally dried under high vacuum which afforded 4r as a yellow semi-solid
(3.8 g, crude). This was then used in the next step without any further purification. LCMS(ESI) m/z
+
91.30 [M+H ]; 84.60% (purity).
1
4.5.29 5-Cyclopentyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4s). Ethyl 3-cyclopentyl-3-oxopropanoate
(2.0 g, 10.8 mmol) was taken in AcOH (10 mL) in a 50 mL round bottom flask under N . To it was added
2
o
H-1,2,4-triazol-5-amine (1.08 g, 13.0 mmol). The reaction mixture was heated at 110 C for 12 h. The
1
reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent and triturated with
diethyl ether. This was finally dried under high vacuum which afforded 4s as a brown solid (1.1 g, 50%).
+
This was then used in the next step without any further purification. LCMS(ESI) m/z 205.05 [M+H ];
66.67% (purity).
4.5.30 5-Cyclohexyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4t). Ethyl 3-cyclohexyl-3-oxopropanoate
(800 mg, 4.04 mmol) was taken in AcOH (4 mL) in a 50 mL round bottom flask under N . To it was
2
o
added 1H-1,2,4-triazol-5-amine (407 mg, 4.84 mmol). The reaction mixture was heated at 115 C for 16
h. The reaction mixture was then evaporated to dryness using toluene as an azeotropic solvent and
triturated with diethyl ether. This was finally dried under high vacuum which afforded 4t as an off-white
solid (410 mg, 46%). This was then used in the next step without any further purification. LCMS(ESI)
+
m/z 217.30 [M-H ]; 52.65% (purity).
4.5.31 7-Chloro-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidine (5a). To
a
solution of 5-phenyl-
[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 4a (10.0 g, 47.2 mmol) was added POCl (44 mL, 472.0 mmol) at 0
3
o o
C. The reaction mixture was then heated at 120 C and monitored by TLC analysis (Hexane / EtOAc =
1:1). The reaction mixture was concentrated using toluene as an azeotropic solvent and quenched with ice
cooled saturated NaHCO solution (20 mL) to pH 8. It was extracted with EtOAc (3 x 50 mL). The
3

2 4
combined organic layer was dried over anhydrous Na SO and concentrated under reduced pressure. The
crude was further purified by flash chromatography on silica gel (60-120 mesh) using 15% hexane-EtOAc
1
as eluent to afford 5a as a pale yellow solid (4.2 g, 39%). H NMR (400 MHz, DMSO-d
6
): δ 8.77 (s, 1H),
+
.38 (s, 1H), 8.32-8.34 (m, 2H), 7.60-7.61 (m, 3H). LCMS(ESI) m/z 231.03 [M+H ]; 92.15% (purity).
8
4.5.32 7-Chloro-5-(4-methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (5b). To a solution of 5-(4-
methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 4b (500 mg, 2.1 mmol) was added POCl
3
(1.6 mL,
6.8 mmol) at 0 C. The reaction mixture was then heated at 100 C and monitored by TLC analysis
Hexane / EtOAc = 1:1). The reaction mixture was concentrated using toluene as an azeotropic solvent
and quenched with ice cooled saturated NaHCO solution (20 mL) to pH 8. It was extracted with EtOAc
3 x 30 mL). The combined organic layer was dried over anhydrous Na SO and concentrated under
o
o
1
(
3
(
2
4
reduced pressure. The crude was further purified by flash chromatography on silica gel (60-120 mesh)
using 5% hexane-EtOAc as eluent to afford 5b as a brown solid (210 mg, 39%). LCMS(ESI) m/z 261.15
+
M+H ]; 85.81% (purity).
[
4
.5.33 7-Chloro-5-(2-methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (5c). To a solution of 5-(2-
methoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 4c (500 mg, 2.1 mmol) was added POCl (5 mL,
2.5 mmol) at 0 C. The reaction mixture was then heated at 100 C and monitored by TLC analysis
Hexane / EtOAc = 1:1). Upon completion, the reaction mixture was concentrated using toluene as an
azeotropic solvent and quenched with ice cooled saturated NaHCO solution (20 mL) to pH 8. It was
extracted with EtOAc (3 x 100 mL). The combined organic layer was dried over anhydrous Na SO and
3
o
o
5
(
3
2
4
concentrated under reduced pressure to afford 5c. This was then used in the next step without any further
+
purification. LCMS(ESI) m/z 261.0 [M+H ]; 53.5% (purity).
4.5.34 7-Chloro-5-(p-tolyl)-[1,2,4]triazolo[1,5-a]pyrimidine (5d). To a solution of 5-(p-tolyl)-
o
(20 mL, 211 mmol) at 0 C.
[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 4d (1.5 g, 6.6 mmol) was added POCl
3
o
The reaction mixture was then heated at 80 C and monitored by TLC analysis (Hexane / EtOAc = 1:1).
Upon completion, the reaction mixture was concentrated using toluene as an azeotropic solvent and
3
quenched with ice cooled saturated NaHCO solution (20 mL) to pH 8. It was extracted with EtOAc (3 x

2 4
100 mL). The combined organic layer was dried over anhydrous Na SO and concentrated under reduced
pressure. The crude was further purified by flash chromatography on silica gel (60-120 mesh) using 20%
+
hexane-EtOAc as eluent to afford 5d as a yellow solid (800 mg, 49%). LCMS(ESI) m/z 245.08 [M+H ];
76.82% (purity).
4.5.35 7-Chloro-5-(4-ethylphenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (5e). To a solution of 5-(4-
3
ethylphenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 4e (400 mg, 1.7 mmol) was added POCl (5 mL, 52.7
o
mmol) at 0 C. The reaction mixture was then heated at 100 C and monitored by TLC analysis (30%,
o
EtOAc-hexane). Upon completion, the reaction mixture was concentrated using toluene as an azeotropic
solvent and quenched with ice cooled saturated NaHCO
3
solution (20 mL) to pH 8. It was extracted with
SO and concentrated
EtOAc (3 x 25 mL). The combined organic layer was dried over anhydrous Na
2
4
under reduced pressure. The crude was further purified by flash chromatography on silica gel (60-120
mesh) using 12% EtOAc-hexane as eluent to afford 5e as a pale yellow solid (250 mg, 58%). LCMS(ESI)
+
m/z 259.44 [M+H ]; 91.33% (purity).
4
.5.36 7-Chloro-5-(4-chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (5f). To a solution of 5-(4-
chlorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 4f (550 mg, 2.2 mmol) was added POCl (2.5 mL,
6.4 mmol) at 0 C. The reaction mixture was then heated at 100 C and monitored by TLC analysis
100% EtOAc). The reaction mixture was concentrated using toluene as an azeotropic solvent and
quenched with ice cooled saturated NaHCO solution (20 mL) to pH 8. It was extracted with EtOAc (3 x
0 mL). The combined organic layer was dried over anhydrous Na
3
o
o
2
(
3
3
2
SO
4
and concentrated under reduced
1
pressure. The crude was triturated with diethyl ether to afford 5f as an off-white solid (310 mg, 52%). H
6
NMR (400 MHz, DMSO-d ): δ 8.78 (s, 1H), 8.42 (s, 1H), 8.35 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.0 Hz,
2
H).
.5.37 7-Chloro-5-(4-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (5h). To a solution of 5-(4-
(6.0 mL, 62.4
4
fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 4h (1.1 g, 4.8 mmol) was added POCl
3
o
mmol) at 0 C. The reaction mixture was then heated at 100 C and monitored by TLC analysis (100%
o
EtOAc). The reaction mixture was concentrated using toluene as an azeotropic solvent and quenched with

ice cooled saturated NaHCO
3
solution (20 mL) to pH 8. It was extracted with EtOAc (3 x 30 mL). The
SO and concentrated under reduced pressure. The
combined organic layer was dried over anhydrous Na
2
4
crude was triturated with diethyl ether to afford 5h as a yellow solid (635 mg, 53%). LCMS(ESI) m/z
+
49.07 [M+H ]; 75.08% (purity).
2
4.5.38 7-Chloro-5-(2-fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (5i). To a solution of 5-(2-
3
fluorophenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 4i (800 mg, 3.5 mmol) was added POCl (10 mL, 112
o
mmol) at 0 C. The reaction mixture was then heated at 100 C and monitored by TLC analysis (30%,
o
EtOAc-hexane). Upon completion, the reaction mixture was concentrated using toluene as an azeotropic
solvent and quenched with ice cooled saturated NaHCO
3
solution (30 mL) to pH 8. It was extracted with
SO and concentrated
EtOAc (3 x 50 mL). The combined organic layer was dried over anhydrous Na
2
4
under reduced pressure. The crude was further purified by flash chromatography on silica gel (60-120
mesh) using 14% EtOAc-hexane as eluent to afford 5i as a pale yellow solid (100 mg, 11%). LCMS(ESI)
+
m/z 248.90 [M+H ]; 50% (purity).
4.5.39 7-Chloro-5-(4-(trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidine (5j). To a solution of
5
-(4-(trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 4j (3.0 g, 10.7 mmol) was added POCl
3
o
o
(
20 mL, 214 mmol) at 0 C. The reaction mixture was then heated at 100 C and monitored by TLC
analysis (50%, EtOAc-hexane). Upon completion, the reaction mixture was concentrated using toluene as
an azeotropic solvent and quenched with ice cooled saturated NaHCO solution (20 mL) to pH 8. It was
extracted with EtOAc (3 x 25 mL). The combined organic layer was dried over anhydrous Na SO and
3
2
4
concentrated under reduced pressure. The crude was further purified by flash chromatography on silica
gel (60-120 mesh) using 13% EtOAc-hexane as eluent to afford 5j as a pale yellow solid (1.75 g, 55%).
+
LCMS(ESI) m/z 299.08 [M+H ]; 93.67% (purity).
4
.5.40 7-Chloro-5-(pyridin-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidine (5k). To a solution of 5-(pyridin-2-
yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 4k (4.0 g, 18.8 mmol) was added POCl (18 mL, 188 mmol) at 0
C. The reaction mixture was then heated at 100 C and monitored by TLC analysis (Hexane / EtOAc =
:1). Upon completion, the reaction mixture was concentrated using toluene as an azeotropic solvent and
3
o
o
1