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Antitubercular and Antiparasitic 2‑Nitroimidazopyrazinones with

Improved Potency and Solubility

Chee Wei Ang, Lendl Tan, Melissa L. Sykes, Neda AbuGharbiyeh, Anjan Debnath, Janet C. Reid,

Nicholas P. West, Vicky M. Avery, Matthew A. Cooper, and Mark A. T. Blaskovich *

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ABSTRACT: Following the approval of delamanid and pretoma-

nid as new drugs to treat drug-resistant tuberculosis, there is now a

renewed interest in bicyclic nitroimidazole scaﬀolds as a source of

therapeutics against infectious diseases. We recently described a

nitroimidazopyrazinone bicyclic subclass with promising antituber-

cular and antiparasitic activity, prompting additional eﬀorts to

generate analogs with improved solubility and enhanced potency.

The key pendant aryl substituent was modiﬁed by (i) introducing

polar functionality to the methylene linker, (ii) replacing the

terminal phenyl group with less lipophilic heterocycles, or (iii)

generating extended biaryl side chains. Improved antitubercular

and antitrypanosomal activity was observed with the biaryl side

chains, with most analogs achieved 2- to 175-fold higher activity

than the monoaryl parent compounds, with encouraging improvements in solubility when pyridyl groups were incorporated. This

study has contributed to understanding the existing structure−activity relationship (SAR) of the nitroimidazopyrazinone scaﬀold

against a panel of disease-causing organisms to support future lead optimization.

tions.⁶The compound was granted conditional approval by the

INTRODUCTION

■

European Medicines Agency (EMA) in 2014 for the treatment

Diseases caused by protozoans and bacteria remain a major

global health threat, especially in low- and medium-income

countries where they aﬀect millions of lives. Current treatment

options are inadequate, attributed to issues such as toxicity,

of multidrug-resistant TB.⁹Nitroimidazole 2 was recently

approved by the FDA in August 2019 as part of an all-oral

combination regimen to treat drug-resistant TB, alongside

bedaquiline and linezolid.¹⁰

low eﬃcacy, high cost, and rapid emergence of drug

Drug repurposing or repositioning programs have opened a

resistance.^1,2Therefore, new and eﬀective therapeutics are

new avenue for nitroimidazoles to target other neglected

urgently needed for the antibiotic and anti-protozoan drug

diseases where R&D funding is limited.¹¹Both enantiomers of

discovery pipelines. Nitroimidazoles were discovered in the

2 were shown to be active against Leishmania donovani, a

early 1950s.³They have broad-spectrum activity across

causative agent of visceral leishmaniasis (VL).¹²However the

parasites, mycobacteria, and both Gram-positive and Gram-

R enantiomer surpassed the activity of its S counterpart in

negative bacteria.³Recently, there has been a great interest in

leishmania-infected macrophages and murine models.¹²

A

developing nitroimidazoles based on new bicyclic core

scaﬀolds, resulting in the success of a nitroimidazooxazole,

delamanid 1 and a nitroimidazooxazine, pretomanid 2 (Figure

1) as approved drugs for treatment of tuberculosis (TB).^4,5

Both 1 and 2 are pro-drugs that share an interesting dual mode

of action. Under aerobic conditions, they have inhibitory

activity against the biosynthesis of mycolic acid, a key

component of the mycobacterial cell wall,^6,7while under

hypoxic conditions, they release nitric oxide intracellularly,

causing respiratory poisoning that kills the non-replicating

bacteria.⁸Nitroimidazole 1 was found to have excellent

bactericidal and sterilizing activity, with MIC values of

0.006−0.024 μg/mL against both drug-susceptible and drug-

resistant Mycobacterium tuberculosis under normoxic condi-

study of 1 in L. donovani revealed its superior activity against

intracellular amastigotes, with >30-fold improvement of EC₅₀

compared to the standard drug miltefosine.¹³Another

nitroimidazooxazole, DNDI-VL-2098 3 that was identiﬁed by

the Drugs for Neglected Diseases initiative (DNDi), also

demonstrated good eﬃcacy in acute and chronic rodent

Received: August 5, 2020

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Figure 1. Bicyclic nitroimidazoles with antitubercular or antiparasitic activity.

models of VL after oral dosing.¹⁴However, 3 was discontinued

from further studies due to safety issues.¹⁵Following the

termination of 3, a novel 7-substituted nitroimidazooxazine,

DNDI-0690 (4) was discovered within a backup program of 2

by the TB Alliance and the University of Auckland. This

preclinical candidate was found to possess excellent in vitro

activity against both L. donovani and Leishmania infantum as

well as displaying a better safety proﬁle than 3; hence, it was

selected for further development.¹⁶Another nitroimidazoox-

azine, DNDI-8219 (5) was discovered from a library of ∼900

pretomanid analogs, with broad-spectrum activity against a

range of reference strains and clinical isolates of Leishmania

while displaying favorable pharmacokinetic proﬁle.¹⁷Similar

programs were expanded to other kinetoplastids, revealing a

new thiazine oxide analog 6 that showed a complete cure in a

mouse model of Trypanosoma brucei brucei infection, when

dosed orally.¹⁸A new nitroimidazolethiazole 7 with potent

activity against Trypanosoma cruzi, the causative agent of

Chagas disease, was also identiﬁed, although it was not active

against L. infantum.¹⁹This suggests the utility of testing

diﬀerent subclasses of bicyclic nitroimidazoles for activity in

various parasite species as well as the possibility to improve

their selectivity against a speciﬁc organism.

The market approvals of nitroimidazoles are encouraging,

but there is still room for improvement. Delamanid 1 suﬀers

from poor water solubility and low absorption, requiring twice-

daily dosing for the treatment of multidrug-resistant TB.²⁰

Pretomanid 2 has better PK properties with daily dosing, but it

is less potent compared to 1. A second-generation nitro-

imidazooxazine, TBA-354 8, was reported to have superior

antitubercular activity than 2 and better metabolic stability

than 1,²¹but it was discontinued from phase I clinical trials due

to neurotoxicity.²²To address some of these drawbacks,

various strategies have been applied, mostly focusing on the

modiﬁcation of the aryl side chain of the imidazo-oxazole/

oxazine ring.²³⁻²⁵A library of new analogs of 2 with extended

side chains and diﬀerent biaryl linkers was synthesized by

Palmer et al. to enhance the solubility and oral activity in

chronic M. tuberculosis infection.²³Most of the lipophilic and

polar linkers showed comparable or improved in vitro

antitubercular activity, whereas the introduction of hydrophilic

groups and replacement of phenyl with pyridine rings were

beneﬁcial in improving the aqueous solubility.²³Similar

measures were also employed by Yempalla et al.²⁴and

Thompson et al.²⁵to enhance the physicochemical/pharmaco-

logical properties of nitroimidazooxazole against M. tuberculosis

and L. infantum, respectively.

Our previous work identiﬁed a new bicyclic nitroimidazole

scaﬀold with promising antitubercular and antiparasitic activity,

namely, nitroimidazopyrazinone 9.²⁶This scaﬀold was derived

from the 4(5)-nitroimidazoles that were previously devel-

oped,²⁷taking inspiration from both metronidazole and 2. The

O-alkylated nitroimidazopyrazine regioisomers of 9 were also

isolated during the reaction, but they were inactive against M.

tuberculosis, even though they retained antiparasitic activity

against Giardia lamblia and T. b. brucei.²⁶This demonstrated

the signiﬁcance of the bicyclic core in determining their

selectivity proﬁle toward diﬀerent organisms. Similar to other

bicyclic nitroimidazoles such as 1 and 2, most of the potent

nitroimidazopyrazinones displayed poor aqueous solubility,

with a few exceptions for some of the actives against T. b.

brucei.²⁶This led us to the current study, in which we have

now designed a new series of analogs in an attempt to improve

solubility without compromising the activity against a panel of

disease-causing organisms. We investigated three approaches,

all focused on modiﬁcation of the substituent at R¹without

B

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Figure 2. Strategy to improve solubility of nitroimidazopyrazinones.

Scheme 1. Synthesis of Nitroimidazopyrazinones 17a−k

a

(i) Various amines, TEA, DCM, 0 °C → rt; (ii) H₂SO₄/HNO₃, 60 °C; (iii) oxalyl chloride, catalytic DMF, DCM, 0 °C → rt, then concentrated

NH₄OH, 0 °C → rt; (iv) bromoacetaldehyde diethyl acetal, K₂CO₃, μW 180 °C; (v) 2 M HCl/1,4-dioxane, μW 120 °C; (vi) various alkyl

bromides, Cs₂CO₃or K₂CO₃, DMF, μW 80−100 °C; (vii) K₂CO₃, MeOH, rt; (viii) various benzyl bromides, NaH, DMF, 0 °C → rt. ^aReported in

a previous study.²⁶

altering the bicyclic imidazopyrazinone core (Figure 2).

Modiﬁed compounds were tested for antibacterial, antiproto-

zoal, and antifungal activity in an eﬀort to identify hit

compounds against these organisms. We chose not to impart

any substitutions at R²and R³as these modiﬁcations were

previously shown to be less favorable for antitubercular

activity, despite showing tolerable potency against T. b.

brucei.²⁶In the ﬁrst approach, a small set of analogs was

prepared where the linker was modiﬁed by introducing ketone,

amide, and ether functionalities with varying lengths to the

benzylic methylene moiety. In the second approach, less

lipophilic heterocycles were used to replace the phenyl group.

Lastly, extended side chains such as biphenyl analogs and their

bioisosteres were synthesized, with and without the incorpo-

ration of less lipophilic heterocycles.

component without altering the active aromatic moiety. The

key nitroimidazopyrazinone intermediate 16 was ﬁrst synthe-

sized in four steps as reported,²⁶starting from the

commercially available imidazo-carboxylic acid 12 (Scheme

1). These involved nitration using concentrated H₂SO₄and

HNO₃, formation of nitroimidazole carboxamide 14, alkylation

with bromoacetaldehyde diethyl acetal under basic conditions,

and ﬁnally cyclization between 1′ imidazole and 2′ free amide

nitrogen to aﬀord the bicyclic core 16.²⁶Diﬀerent linker

variants (17a−g) were synthesized from 16 through alkylation

with alkyl halides using basic carbonate (Cs₂CO₃or K₂CO₃) as

a catalyst. Ketone-linked products (17a−c) were synthesized

from bromoacetophenone derivatives at room temperature,

though with poor yield (23−41%). For amide linker variants

(17d−f), intermediate bromo-N-substituted acetamides (11a-

c) were ﬁrst synthesized via acylation of various amines and 2-

bromoacetyl bromide in the presence of trimethylamine²⁸

followed by N-alkylation under microwave irradiation.

Hydroxyl derivative (17h) was prepared from 17g by

deacetylation using K₂CO₃in methanol, which was then

CHEMISTRY

■

From our previous SAR ﬁndings, aromatic substituents at R¹

were preferred over polar side chains and bulky aliphatic

groups.²⁶Therefore in series 1, we sought an alternative

approach to reduce the lipophilicity by varying the linker

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Scheme 2. Synthesis of Nitroimidazopyrazinones 26a−k

a

(i) SOCl₂, DCM, 0 °C → rt; (ii) PBr₃, DCM, 0 °C → rt; (iii) 4 N HCl, chloroacetic acid, reﬂux; (iv) 18−21, 24−25, K₂CO₃, DMF, μW 80−100

°C; (v) bromoacetaldehyde diethyl acetal, K₂CO₃, DMF, μW 150 °C; (vi) 1 M HCl, dioxane, μW 120 °C. Intermediates 19 and 20 were

synthesized using the same method as 18.

coupled with the desired benzylic bromides in the presence of

NaH to form the ether-linked analogs (17i−k).

ﬁltration after neutralizing with NaHCO₃. Subsequently, N-

alkylation was performed between 16 and the halide

intermediates to aﬀord the ﬁnal products (26a, c−f, h) in

42−75% yield after puriﬁcation. Alternatively, 26b, g, i−k were

prepared via route 2, which involved the formation of 4(5)-

nitroimidazole carboxamides 27b, g, i−k of diﬀerent

derivatives.²⁷Alkylation of 27b, g, i−k with bromoacetalde-

hyde diethyl acetal under microwave conditions provided 28b,

g, i−k followed by cyclization in acid to give the bicyclic

products in 13−83% yield.

For series 2, less lipophilic aromatic rings with hydrogen

bond donors/acceptors, such as pyridine, thiazole, and

imidazole were introduced. Two diﬀerent routes, as described

in Scheme 2, were utilized to synthesize 26a−k. In the ﬁrst

route, the chloride intermediates 18−20 were converted from

the alcohol derivatives in thionyl chloride,²⁹whereas 21 was

formed by bromination using phosphorus tribromide.³⁰These

halide intermediates were used immediately in the following

step due to their instability. Benzimidazoles 24 and 25 were

made from 22 and 23 and chloroacetic acid by the Philip

method,³¹which were easily isolated in high purity by vacuum

For the third approach, a variety of biaryl side chain

intermediates (33b−m) were synthesized to produce the series

3 analogs 34b−m, while biphenyl 34a was prepared from

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Scheme 3. Synthesis of Nitroimidazopyrazinones with Biaryl Side Chain 34a−p³²⁻³⁴

a

(i) Pd(PPh₃)₄, K₂CO₃, THF, rt → 80 °C, N₂; (ii) NaBH₄, EtOH, rt; (iii) thionyl chloride, DCM, 0 °C → rt; (iv) K₂CO₃, DMF, μW 80−100 °C;

(v) nitroimidazole acid chlorideintermediate,²⁷TEA, DCM, 0 °C → rt; (vi) bromoacetaldehyde diethyl acetal, K₂CO₃, DMF, μW 150 °C; (vii) 1

M HCl, dioxane, μW 120 °C. Intermediate 33a was from a commercial source.

commercial source 33a (Scheme 3). Aldehydes 29a−c were

ﬁrst reacted with boronic acids 30a−e via palladium-catalyzed

Suzuki coupling to form 31b−m,³²which were then puriﬁed

by silica chromatography. Reduction of 31b−m using sodium

borohydride produced alcohols 32b−m under mild conditions

(room temperature) and good yield. Chlorination under

treatment of thionyl chloride furnished 33b−m, which were

used directly in the ﬁnal step of alkylation. Synthesis of 34n,o

was also accomplished using similar chemistry, starting from

the reduction of aldehydes 31n,o. While the isolation yield of

these extended biaryl analogs were varied (37−98%), they

were synthesized rapidly (less than 1 h) under microwave

conditions and less impurities were observed by LC-MS.

Compound 34p was synthesized from 4(5)-nitroimidazole

carboxamide 36 through acylation with (4-(piperidin-1-yl)-

phenyl)methanamine 35 followed by N-alkylation and

cyclization under a similar reaction condition as 27b, g, i−k.

RESULTS AND DISCUSSION

■

A total of 36 new nitroimidazopyrazinones (17a−f, 17i−k,

26a−k, and 34a−p) and 2 previously reported analogs (17g,h)

were screened for in vitro activity against a wide range of

disease-causing organisms. These include M. tuberculosis grown

under normoxic and hypoxic conditions and T. b. brucei, a

surrogate species for Human African trypanosomiasis (HAT).

Following the potential of nitroimidazopyrazinones shown

against T. b. brucei from our earlier investigation,²⁶we have

also expanded our study to another pathogenic kinetoplastid,

Trypanosoma cruzi, which causes Chagas disease. We

hypothesized that these new nitroimidazopyrazinones might

possess activity against Giardia lamblia and Entamoeaba

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Table 1. Physicochemical Properties and Antimycobacterial and Antiparasitic Activities of 17a−k Compared to the Parent

g

Analogs 16a−d

a

b

AlogP values were calculated using Pipeline Pilot (Accelrys, version 9.1.0.13). Solubility in water and PBS (pH 7.4) were determined by LC-UV

c

(254 nm). H37RV, Mtb-O₂primary screen at 32 μg/mL n = 2, Mtb-hypoxia primary screen at 32 μg/mL n = 2, MIC-normoxia/hypoxia of active

compounds n = 3. Isoniazid control Mtb-normoxia MIC = 0.04 μg/mL, Mtb-hypoxia MIC > 5 μg/mL. Control pentamidine IC₅₀= 0.002 μM,

diminazene aceturate IC₅₀= 0.062 μM, puromycin IC₅₀= 0.05 μM, n = 2. Control nifurtimox IC₅₀= 1.4 μM, benznidazole IC₅₀= 5.2 μM,

puromycin IC₅₀= 2.5 μM, posaconazole IC₅₀= 0.04 μM, n = 2. ATCC CRL-1573, CC₅₀n = 3. Additional cytotoxicity data are detailed in the

d

e

f

g

Supporting Information, Table S2. For compounds that did not completely inhibit growth at the concentration tested, the percentage inhibition

(% I) is given. ND, not determined.

histolytica as their ﬁrst generation and the monocyclic

intermediates, nitroimidazole carboxamides,²⁷were active

against these intestinal parasites. Therefore, selected com-

pounds from each series were screened for activity against

these protozoans. Given other nitroimidazoles (e.g., metroni-

dazole) have antibacterial activity beyond M. tuberculosis, we

assayed their antibacterial properties against representative

ESKAPE pathogens (Escherichia coli, Staphylococcus aureus,

Klebsiella pneumoniae, Pseudomonas aeruginosa, and Actineto-

bacter baumannii) as well as their antifungal potential against

Cryptoccoccus neoformans and Candida albicans were assessed.

Mammalian cell viability was evaluated against human

embryonic kidney (HEK293) cells in order to establish a

preliminary selectivity index for antimicrobial activity versus

mammalian toxicity. For compounds tested against T. cruzi,

cytotoxicity against 3T3 host cells was also determined.

Lipophilicities of these analogs were estimated using Pipeline

Pilot (Accelrys, version 9.1.0.13). Solubility was measured in

water and phosphate-buﬀered saline (PBS), pH 7.4 using

HPLC with UV analysis. Representative compounds with weak

base functionalities were also tested for their aqueous solubility

in acetate buﬀer, pH 4.5 and 0.1 M HCl, pH 1. The low pH

mimics the gastric ﬂuid environment, which is beneﬁcial to

enhance dissolution of weakly basic drugs.³⁵

17e, and 17i were inactive (MIC_normoxia≥ 32 μg/mL),

although 17i showed ∼11-fold of improvement in its solubility.

Some activity was restored with the addition of an electron-

donating 4-methyl group at the phenyl ring. However, this

more polar series generally showed unfavorable antimycobac-

terial activity (Table 1). As many of the recently developed

anti-TB agents are highly lipophilic, this suggests the

importance of lipophilicity in a molecule to penetrate into

the hydrophobic phase of mycobacteria cell wall.³⁶

Determination of antitrypanosomal activity disclosed a

diﬀerent SAR. Amide linkers were overall well tolerated with

respect to T. b. brucei activity, giving at least an 8-fold

improvement of activity compared to the methylene linker

(incomplete inhibition at 16 and 40 μM). Compound 17d

(−CH₂CONH−) with an amide bond directly linked to the

phenyl side chain (IC₅₀= 0.86 μM) was ∼3-fold more potent

than 17e (−CH₂CONHCH₂−) with an extra carbon length

(IC₅₀= 2.4 μM). Addition of a 3-triﬂuoromethoxy group at the

phenyl ring was also favorable (∼7-fold, IC₅₀= 0.36 μM).

Though the presence of amide linker lowered the calculated

lipophilicity (ΔAlogP ≈ −0.94 units), there was little or no

improvement in their aqueous solubility. The introduction of a

more soluble ether group linker displayed only a mild eﬀect on

T. b. brucei activity compared to the parent analogs (16a−c).

These ether-linked derivatives 17i−k were the only actives in

this series against T. cruzi, with IC₅₀values ranging from 1.3 to

2.3 μM, comparable activity to one of the standard drugs

nifurtimox (IC₅₀= 1.4 μM) and more potent than

benznidazole (IC₅₀= 5.2 μM). Though they were less active

than the parent analog 16b, these series demonstrated

improved solubility in some cases and had good selectivity

indices of >43 to >77.

Series 1 (Table 1). For the ﬁrst series 17a−k, insertion of

ketone, amide, and ether groups to the methylene chain led to

overall reduced lipophilicity based on Pipeline Pilot AlogP

calculations (Table 1). The presence of oxygen in ether and

ketone linked molecules functions as a strong hydrogen bond

acceptor, whereas the amide linker serves as a strong hydrogen

bond acceptor and as a modest hydrogen bond donor.

Compared to our reported analog 16a with a MIC of 0.5−1

μg/mL against H37Rv strains under aerobic growth, 17a, 17d,

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g

Table 2. Physicochemical Properties and Antimycobacterial and Antiparasitic Activities of 26a−k

a

b

c

AlogP values were calculated using Pipeline Pilot (Accelrys, version 9.1.0.13). Solubility were determined by LC-UV (254 nm). H37RV, Mtb-O₂

primary screen at 32 μg/mL n = 2, Mtb-hypoxia primary screen at 32 μg/mL n = 2, MIC-normoxia/hypoxia of active compounds n = 3−4. For

compounds that gave varied MIC in diﬀerent replicates (n = 3−6), a median value was reported with the MIC range indicated in parentheses.

d

Isoniazid control Mtb-normoxia MIC = 0.04 μg/mL, Mtb-hypoxia MIC > 5 μg/mL. Control pentamidine IC₅₀= 0.002 μM, diminazene aceturate

e

IC₅₀= 0.062 μM, puromycin IC₅₀= 0.05 μM, n = 2. Control nifurtimox IC₅₀= 1.4 μM, benznidazole IC₅₀= 5.2 μM, puromycin IC₅₀= 2.5 μM,

f

posaconazole IC₅₀= 0.04 μM, n = 2. ATCC CRL-1573, CC₅₀n = 3. Additional cytotoxicity data are detailed in the Supporting Information, Table

g

S2. For compounds that did not completely inhibit growth at the concentration tested, the percentage inhibition (% I) is given. ND, not

determined.

Series 2 (Table 2). Overall, compounds with less lipophilic

heterocycles replacing the aryl substituent displayed signiﬁcant

improvement of solubility compared to the phenyl substituent.

However, in most cases, their antimycobacterial activity was

compromised. For example, compound 26a containing a

thiazol-5-yl substituent was >50-fold more soluble than 16a,

though its activity against M. tuberculosis under normoxic

conditions was reduced by 32-fold (Table 2). Replacement of

the phenyl ring with a more soluble pyridine group was also

detrimental to its activity. However, addition of a 4-CF₃

electron-withdrawing group managed to restore some of the

activity (MIC_normoxia= 2 μg/mL cf. MIC_normoxiaof 16a = 0.5−1

μg/mL) and concurrently provided superior aqueous solubility

(30 μM cf. 16a: 3.1 μM). It was found that compounds with

weak basic nature of 26j allowed for further improvement of

solubility under acidic conditions (70 μM at pH 4.5 and > 200

μM at pH 1). It is noted that 26j was also active against T.

cruzi at submicromolar concentrations (IC₅₀= 0.54 μM), and

selectivity index of >136 to 3T3 host cells (Supporting

Information, Table S2). As this analog was moderately active

against M. tuberculosis (MIC_normoxia= 4 μg/mL), this indicates

the possibility to identify compounds with broad-spectrum

activity and low toxicity against mammalian cells that could

potentially treat both tuberculosis and kinetoplastid infections.

Series 3 (Table 3). Following the precedent set by several

new bicyclic nitroimidazoles with biaryl side chains such as 4

and 8 that are in pre-clinical development against leishmaniasis

21

and tuberculosis, respectively,^16,a similar strategy was

negative AlogP values generally possessed poor MIC_normoxia

≥

employed in the third series. As suggested from the SAR for

series 1 and 2, compounds with less lipophilicity had less

favorable antimycobacterial activity. Therefore, extended and

highly lipophilic biphenyl side chains were explored to

understand whether this property is the main driver. It was

also postulated that the extended side chains could interact

more eﬃciently with the hydrophobic pocket in Ddn, the

nitroreductase enzyme responsible for the bioactivation of

bicyclic nitroimidazoles 1 and 2.³⁷To counter the solubility

issue, bioisosteres were also introduced to replace one of the

phenyl rings in this series. The eﬀect of diﬀerent biaryl

geometries with 4′ and 3′-linked biaryls as well as the position

of the substituents were investigated. Based on the results of

the previously developed monoaryl series, we included the

16 μg/mL. Analogs with positive AlogP in this series remained

active (MIC_normoxia= 2−4 μg/mL), with the exception of

compound 26f that displayed solubility problems even in the

DMSO stock solution. None of the active analogs surpassed

the activity of the parent compounds (for example, 16a−d),

suggesting that an alternative strategy is required to achieve a

desirable compromise between solubility and bioactivity.

We next investigated the SAR of this series against T. b.

brucei. As in series 1, in most cases, the less lipophilic

heterocycles were well tolerated, with an IC₅₀of <10 μM.

Compound 26j with a quinolone substituent was the most

active in this series (IC₅₀= 0.89 μM) and gave a substantial

enhancement of solubility (>8-fold) compared to 16a. The

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Figure 3. Inﬂuence of heterocyclic rings on the bioactivity and solubility of nitroimidazopyrazinones.

most active substituents, 4-triﬂuoromethoxy, 4-methyl, and 3-

triﬂuoromethoxy, on the terminal ring.

point by 100 °C will change solubility by 10-fold.³⁹Hence, an

alternative strategy was attempted by disrupting their

molecular symmetry. The crystal packing of a molecule is

inﬂuenced by its molecular symmetry and planarity, and

improved solubility can be achieved by decreasing the

eﬃciency of its crystal packing to lower the melting point.⁴⁰

In the 3′-linked phenylpyridines 34k−m, we have observed at

least 3- to >16-fold of improvement in solubility at pH 1

compared to 4′-linked phenylpyridines, with the 3′-substitu-

tion disrupting the potential for planar packing. Compounds

34k−m also showed 2- to 128-fold better antitubercular

activity compared to their 4′-linked analogs. Among them, 34l

displayed an MIC of 0.0625−0.125 μg/mL, which was

equipotent with the parent analog 16c. As opposed to the

4′-linked biaryls, the para-substitution (34k) was preferred

over its meta group (34m) in terms of potency and solubility.

Swapping the positions of the less lipophilic ring in the biaryl

system led to substantial eﬀects on potency and physicochem-

ical properties (Figure 3). Biaryl compound 34e with a

terminal 3-pyridine ring displayed 64-fold of improvement in

antitubercular activity under aerobic conditions as compared to

34f with a terminal phenyl ring. Compound 34e was also more

soluble than 34f in both water and PBS, pH 7.4, and its

solubility was further improved at pH 4.5 (19 μM) and pH 1

(>200 μM). When pyridine was replaced by a thiazole group at

the terminal ring (34n), there was a complete loss of activity,

indicating that the antitubercular activity was strongly

dependent on the nature of the heterocycles. In spite of

having similar AlogP values, 34n was also at least 3-fold less

soluble than 34e in water and PBS. When the position between

thiazole and phenyl rings was inverted (34o), the anti-

tubercular activity improved at least 32-fold, reaching an

MIC_normoxiaof 0.5−1 μg/mL. Removing the aromaticity by

replacing the pyridine ring with a piperidine ring (34p)

resulted in 2- to 4-fold reduction of activity against replicating

Generally, the biphenyl analogs 34a−d from Table 3 show

that these conformationally rigid extended side chains did not

contribute to a better activity against M. tuberculosis. In fact,

they were 16- to 64-fold less active than the monoaryl analogs

16a−d. This observation is diﬀerent from what has been

reported for the biphenyl structures of analogs of pretomanid

2, in which the para-linked compounds retained or improved

their antitubercular activity compared to the monoaryl 2.³⁸

This suggests a diﬀerent binding mode of the nitroimidazopyr-

azinone scaﬀold 9 in the active site compared to 2, which is not

unexpected given the planar nature of 9 compared to 2. There

was a preference for substitution at the meta position over para

at the terminal ring of the 4′ biphenyl analogs. A similar trend

was observed for the less lipophilic phenylpyridine analogs

(ΔAlogP ≈ −0.7 unit), where the 3-triﬂuoromethoxy congener

34i was 8-fold more active than its 4-triﬂuoromethoxy

counterpart. Overall, the phenylpyridines 34f−i gave similar

or 2-fold reduced activity than the 4′-biphenyl analogs 34a−d,

though the solubility (at pH 1) was improved for some of

these derivatives. The position of the nitrogen atom at the

pyridine ring was also found to be important for its bioactivity.

Replacement of the 3-pyridine ring in 34g by 2-pyridine (34j)

substantially improved its antimycobacterial activity, from an

MIC of 32 μg/mL to 0.0625−0.125 μg/mL against the

replicating M. tuberculosis. However when M. tuberculosis was

grown under hypoxic conditions (0.1% oxygen), 34j exhibited

only marginal inhibitory activity, though still better than 34g.

Under acidic conditions (pH 1), 34j possessed >3-fold better

solubility than 34g.

According to the general solubility equation developed by

Jain and Yalkowsky, a compound can exhibit poor solubility

due to high lipophilicity and high stability in the crystalline

state. It is estimated that altering log P by 1 unit or melting

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Table 4. Additional Activity Data for Selected Analogs from Each Series

antiparasitic IC₅₀(μM) (pIC₅₀SE)

antibacterial MIC (μg/mL)

a

b

c

d

compound

G. lamblia

E. histolytica

ESKAPE

antifungal MIC (μg/mL)

pretomanid 2

16a

16b

3.0 (5.5 0.02)

5.0 (5.3 0.05)

3.5 (5.5 0.01)

1.7 (5.8 0.03)

9.3 (5.0 0.03)

>50 (<4.3)

>32

16d

>50 (<4.3)

Series 1

17a

17d

17e

17i

18 (4.7 0.03)

>50 (<4.3)

23 (4.6 0.02)

>50 (<4.3)

>32

17k

Series 2

26a

26b

26j

26k

40 (4.4 0.04)

19 (4.7 0.04)

7.1 (5.1 0.02

2.4 (5.6 0.04)

>50 (<4.3)

>32

Series 3

34a

3d

34k

34p

0.5 (6.3 0.11)

37 (4.4 0.08)

1.8 (5.7 0.09)

5 (5.3 0.03)

>50 (<4.3)

>32

a

b

c

WB. HM1:IMSS. ESKAPE pathogens include Staphylococcus aureus (MRSA ATCC 43300), Escherichia coli (ATCC 25922), Klebsiella

d

pneumoniae (ATCC 700603), Acinetobacter baumannii (ATCC 19606), and Pseudomonas aeruginosa (ATCC 27853). Candida albicans (ATCC

90028) and Cryptococcus neoformans var. grubii H99 (ATCC 208821).

a

Table 5. In Vitro ADME Properties of Representative Compounds

PPB (%)

microsomal stability (% remaining at 2 h)

plasma stability (% remaining at 2 h)

compound

human

mouse

human

mouse

human

mouse

pretomanid 2

17k

26j

34e

34g

34j

34k

34p

97 1.1

78 4.1

79 0.44

96 0.88

>99

97 0.30

91 1.3

74 3.4

72 1.3

93 0.26

>99

99 0.02

97 5.0

8.5 0.73

98 2.5

96 7.7

98 2.6

>99

92 2.7

ND

20 4.0

ND

98 1.9

ND

96 6.9

96 6.8

95 4.3

93 2.6

98 2.1

>99

96 1.9

ND

>99

ND

>99

ND

>99

37 1.7

>99

ND

a

Values are presented as mean of three replicates SD. Microsomal stability control, verapamil at 30 min = 9% (human), 2% (mouse). Plasma

stability control, eucatropine at 2 h = 28% (human), 29% (mouse). % bound of PPB control, sulfamethoxazole = 57% (human), 9% (mouse). ND,

not determined.

M. tuberculosis under aerobic conditions (MIC_normoxia= 1−2

μg/mL) but retained similar activity against non-replicating M.

tuberculosis (MIC_hypoxia= 8 μg/mL).

brucei and T. cruzi, respectively, with 100% activity at E_max

concentrations (maximal % inhibition plateau generated in

dose−response curves). This compound also exhibited

selectivity indices against HEK293 cells (20 h assay) of

>37,000 and >4625, and selectivity of >2288 to 3T3 host cells

(69 h assay, Supporting Information, Table S2). It should be

noted that 34j was one of the most active antitubercular hits,

suggesting a broad-spectrum activity. Altering the position of

the thiazole ring from 34n to 34o enhanced the activity against

both T. b. brucei and T. cruzi, whereas a marginal eﬀect was

observed when swapping pyridine from 34e to 34f. When the

terminal 3-pyridine ring was replaced by a saturated hetero-

cycle (piperidine), the antitrypanosomal activity of 34p was

diminished.

In comparison to the monoaryl compounds, the biaryl series

demonstrated superior activity against both T. b. brucei and T.

cruzi. Of a total of 16 biaryl analogs synthesized, 14 were

identiﬁed as active hits, with mean IC₅₀values of <1 μM and

selectivity indices of >10 for parasitic activity compared to

mammalian cell cytotoxicity. More than half of these hits were

able to achieve IC₅₀at ≤0.1 μM. Contrary to the SAR in

mycobacteria, 4′-phenylpyridines 34f−i possessed better

activity against T. b. brucei than the 4′-biphenyl side chains

34a−d. Compounds 34g-i were also ∼1.1- to 8.4-fold more

active than 3′-linked phenylpyridines 34k−m, with an IC₅₀

range of 0.008−0.026 μM. A similar SAR trend was observed

in T. cruzi, though their activity was less compared to T. b.

brucei in most of the cases. Compound 34j with 2-pyridine ring

was identiﬁed as the most potent candidate, giving an

impressive IC₅₀of 0.002 μM and 0.016 μM against T. b.

Activity against Intestinal Parasites, ESKAPE Patho-

gens, and Fungi. Given the good activity shown by some of

our previously reported nitroimidazopyrazinones against G.

lamblia (IC₅₀= 1.6−3.5 μM),²⁶we ﬁrst investigated

compounds from Series 1 to examine the linker eﬀect. Similar

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to the trends shown in M. tuberculosis, modiﬁcation of the

methylene chain resulted in drastic loss of activity (Table 4).

Replacing the benzyl substituent with less lipophilic mono-

heterocycles (Series 2) also proved to be unfavorable. Fused

rings such as quinolone (26j) and benzodioxole (26k)

managed to restore some of the activity, with IC₅₀values of

7.1 and 2.4 μM, respectively. Most of the tested biaryl analogs

remained active in the primary screen at 50 μM. However,

some inconsistent SAR results were found when determining

their IC₅₀values. The biphenyl compound 34a achieved 10-

CONCLUSIONS

■

Current therapeutics against neglected tropical diseases are

imperfect due to toxicity, inadequate eﬃcacies against all

subspecies/species, and prolonged regimens with poor patient

compliance. M. tuberculosis has developed resistance rapidly,

leading to the emergence of multidrug- and extensively drug-

resistant TB with low cure rates. To improve treatment

regimens in an eﬀort to reduce poor patient compliance, low

dosing frequency, reduced toxicity, and easy administration are

required. Oral, aﬀordable drugs are preferred as they are more

convenient to be used, thus increasing compliance especially in

developing countries where treatment is often far from the

patient. However, many new chemical entities are poorly

soluble, which makes them diﬃcult to achieve desired

therapeutic concentration after oral dosing.

fold better potency than its monophenyl counterpart (IC₅₀

=

0.5 μM cf. IC₅₀of 16a = 5 μM), whereas addition of a 3-OCF₃

substituent (34d) reduced activity by >20-fold. Follow-up

study is hence required in the future to determine the

inhibitory concentration of more analogs from this series.

Besides antigiardial activity, selected analogs from each

series were also screened against E. histolytica, ESKAPE

bacteria strains and two fungal pathogens. Similar to our ﬁrst

generation of nitroimidazopyrazinones, none of the tested

compounds were active against these organisms. We have also

counter screened these compounds against mammalian cell

line HEK293 and minimal reduction in cell viability was seen

up to the highest tested concentrations (>50 or >100 μM,

respectively, depending on the solubility in DMSO stock,

Tables 1−3).

Plasma Protein Binding and Microsomal and Plasma

Stability. To determine the drug-like properties of these new

series, a small subset of compounds was evaluated in vitro to

determine their plasma protein binding (PPB), microsomal

stability, and plasma stability (Table 5). The more soluble

analogs 17k and 26j demonstrated a reduced propensity to

bind to plasma protein (72−79% PPB in human and mouse

plasma) compared to other compounds. The biaryl series were

generally highly bound to plasma, especially compounds 34g,

34j, and 34k that demonstrated >99% PPB due to their high

lipophilicity (AlogP > 3). On the other hand, phenylpyridine

34e and phenylpiperidine 34p, with better solubility and lower

lipophilicity, demonstrated a slightly lower PPB (93−99%). A

good correlation between the compound lipophilicity and PPB

was identiﬁed, suggesting the potential to utilize this parameter

to modulate protein binding. However, it is also known that

free drug concentration is not merely determined by PPB but

also by hepatic intrinsic clearance after oral dosing.⁴¹Thus, we

investigated the metabolic stability of these analogs in human

liver microsomes (HLM). The biaryl compounds tested were

highly stable up to 2 h. Replacing the terminal aryl ring with

piperidine (34p) reduced the microsomal stability from >95%

to 37%. Similar to some of the reported pretomanid analogs,⁴²

benzyl ether substituent (17k) was highly susceptible to

metabolism, with 8.5% of intact compound remaining at 2 h.

We have also expanded the metabolic assay to CD-1 mouse

liver microsomes (MLM) for two hit compounds, 34e and 34j,

prior to assessment in future in vivo studies against T. brucei

and T. cruzi. The most potent compound 34j displayed

excellent stability in both HLM and MLM. However,

interspecies variation was observed for phenylpyridine 34e,

with 20% of intact compound remaining at 2 h in MLM

compared to 96% in HLM. Similar to the ﬁrst generation of

nitroimidazopyrazinones,²⁶these new series remained highly

stable in plasma (>90% at 2 h) regardless of their side chains.

In this study, we aimed to optimize the solubility and activity

proﬁles of a new bicyclic nitroimidazole subclass, namely,

nitroimidazopyrazinone, as an oral treatment against tuber-

culosis and parasitic infections. Three new exploratory series

were synthesized, featuring a range of linker groups, hetero-

cycles, and extended biaryl side chains. Modiﬁcation of the

methylene chain of 16 with polar linkers was not favorable for

activity against M. tuberculosis, though some of them were

active against the trypanosome species tested. Similarly,

attempts to replace the phenyl substituent with less lipophilic

heterocycles resulted in signiﬁcant improvements of solubility

but with compromised antitubercular activity. The series with

biaryl extended side chains provided the most promising

outcome in optimizing both the potency and solubility while

retaining good selectivity index of >10 to mammalian cell lines.

Several phenylpyridine analogs showed improved solubility at

low pH conditions without compromising their activity against

M. tuberculosis. It was also found that the meta-linked analogs

were superior to the more linear, para-linked counterparts, in

terms of their solubility and antimycobacterial activity. The

biaryl compounds were highly active against both T. b. brucei

and T. cruzi, with an increase in activity by 1−4 orders of

magnitude in comparison to the monoaryl series. Compound

34e with a terminal pyridine ring combined both better

solubility proﬁle (>200 μM at pH 1) with good bioactivity

against M. tuberculosis and trypanosomes but displayed a

shorter half-life in mouse microsomes. The 2-pyridine 34j was

identiﬁed as the most potent hit, demonstrating submicromo-

lar to nanomolar inhibitory concentrations against M. tuber-

culosis, T. b. brucei, and T. cruzi while showing low cytotoxicity

against HEK293 cells (CC50 > 74 μM, or selectivity indices of

>510 to >37,000). Compound 34j also possessed good

metabolic stability in both microsomes and plasma, despite

having high PPB levels. As many clinical drugs are highly

bound to plasma, it is not recommended to optimize this

parameter in early-stage drug discovery.

This study highlights the potential of nitroimidazopyrazi-

none as a promising bicyclic subclass to be developed as anti-

infective agents. It was found that some of the potent

antitubercular biaryl compounds were highly active against

both T. b. brucei and T. cruzi, unveiling their broad-spectrum

activity for treating multiple infections including neglected

tropical diseases. Several compounds were also moderately

active against G. lamblia, albeit with no activity against E.

histolytica. Similar to the activity proﬁle of pretomanid 2, none

of these nitroimidazopyrazinones was active against ESKAPE

and fungal pathogens (MIC > 32 μg/mL). For further

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General Procedure for Chlorination of Alcohol. Method D:

Alcohol (1 equiv) was added with DCM (20 vol) and was chilled at 0

°C. Thionyl chloride (2−3 equiv) was added dropwise, and the

reaction was continued to stir at room temperature for 1−24 h. The

mixture was then diluted with NaHCO₃(200 vol) and extracted with

EtOAc (3x). The organic layer was collected and dried over MgSO₄

before removing the volatiles in vacuo.

development of this series, it appears that both broad-spectrum

(antitubercular and antiparasitic) and narrow-spectrum strat-

egies are possible, but it is less clear which approach is most

desirable. Co-infection with both mycobacteria and trypano-

somes happens in some regions of the world, where a broad-

spectrum treatment could be useful. However, this is oﬀset by

the possibility that widespread use against one organism could

inadvertently lead to the development of resistance against the

other. Since nitroimidazoles are pro-drugs that require

bioactivation of their nitro group to exert the activity,

resistance can arise when there is a change of activity or

mutations of the reductive enzymes.³In some cases, this

resistance can be overcome by other nitroimidazoles with

diﬀerent mode of action. Further studies are needed to assess

the cross-resistance proﬁle of the nitroimidazopyrazinones. In

vitro ADME and cytotoxicity studies have revealed favorable

drug-like proﬁles for the hit compounds, which warrant

advancement to in vivo testing to determine pharmacokinetic

proﬁles and assess activity in both acute and chronic infection

models of M. tuberculosis, T. b. brucei, and T. cruzi.

General Procedure for Amide Coupling. Method E: 4-Nitro-1H-

imidazole-2-carboxylic acid (1 equiv) was added with anhydrous

DCM (20 vol) and was chilled at 0 °C under N₂. Oxalyl chloride (2

equiv) was added dropwise over 10 min followed by addition of

catalytic DMF. The reaction was allowed to warm to room

temperature and was continued to stir overnight. Additional oxalyl

chloride and DMF was added to the reaction mixture when necessary.

Volatiles were removed in vacuo, and the excess oxalyl chloride was

removed by co-evaporation with toluene two times. The resulting acid

chloride crude solid (1 equiv) was then added immediately to

anhydrous DCM (15 vol) and triethylamine (2 equiv) in an ice bath.

The blue-purple solution was then added dropwise to the respective

amine (1.2 equiv) in 5 vol of anhydrous DCM at 0 °C under N₂.

General Procedure for Alkylation of Imidazo Carboxamide.

Method F: A mixture of imidazo carboxamide (1 equiv) and

potassium carbonate (3 equiv) in dry DMF (20 vol) was reacted with

bromoacetaldehyde diethyl acetal (1.5 equiv x 2) in a microwave

reactor at 150 °C (15 min x 2). If necessary, the reaction was heated

for another 15 min at 150 °C to consume the starting material.

General Procedure for Ring Closure to Form Imidazopyrazi-

nones. Method G: To a stirring solution of imidazo carboxamide (1

equiv) in 1,4-dioxane (10 vol) was added 2 M aq. HCl (10 vol). The

reaction was reacted at 120 °C under microwave for 20 min.

General Procedure for Suzuki Coupling Reaction. Method H: To

a stirring mixture of 4-bromobenzaldehyde or bromonicotinaldehyde

(1 equiv) in THF was added 3 equiv of potassium carbonate in

distilled water followed by addition of 0.08 equiv of tetrakis-

(triphenylphosphine)palladium (0). The mixture was stirred at room

temperature for 5 min. Boronic acid (1.1 equiv) was then added, and

the reaction was heated at 80 °C for 18 h.

GENERAL EXPERIMENTAL SECTION

■

All the reagents and solvents were obtained from commercial sources

and were used without further puriﬁcation. Progression of reaction

was monitored by TLC using Merck alumina sheets pre-coated with

silica gel 60 F254 and was visualized using a UV lamp. Reactions

requiring anhydrous conditions were performed under an inert

atmosphere of nitrogen. Puriﬁcation of compounds was done using a

Biotage Isolera or Grace Reveleris chromatography system. NMR data

were collected at 298 K on a Varian Unity 400 MHz or Bruker

Advance 600 MHz spectrometer. Chemical shifts were measured

relative to tetramethylsilane or the residual solvent signals (DMSO-

d₆) as the internal reference. Data are presented as follows: chemical

shift (ppm), multiplicity (s = singlet, d = doublet, t = triplet, q =

quartet, m = multiplet, bs = broad singlet) and coupling constant

(Hz). All the peaks for ﬁnal products were assigned based on one-

dimensional and two-dimensional NMR analysis. Analytical LC-MS

was performed on a Shimadzu LCMS-2020 using 0.05% formic acid

in water (solvent A) and 0.05% formic acid in acetonitrile (solvent B)

as the mobile phase. LC-MS condition A: column Zorbax Eclipse

XDB-Phenyl, 3.0 × 100 mm, 3.5 μm; column temperature: 40 °C;

ﬂow: 1 mL/min; gradient timetable: 0.00 min, 5% B; 0.50 min, 5% B;

3.00 min, 100% B; 4.2 min, 100% B; 5.00 min, 5% B. LC-MS

condition B: column: Waters Atlantis T3, 2.1 × 50 mm, 5 μm;

column temperature: 40 °C; ﬂow: 0.75 mL/min; gradient timetable:

0.00 min, 5% B; 0.50 min, 5% B; 3.30 min, 25%B; 3.50 min, 100% B;

4.00 min 100% B; 5.00 min, 0% B. All ﬁnal products were >95% pure

as determined by LC-MS using UV at 254 nm, ELSD, and APCI/ESI-

MS. The purity percentage was indicated by Abs %.

General Procedure for Reduction of Aldehyde to Alcohol.

Method I: Sodium borohydride (1.3 equiv) was added in one portion

to a stirring solution of aldehyde (1 equiv) in 20 vol of ethanol that

was chilled to 0 °C. The mixture was continued to stir at room

temperature for 30 min before the solvent was removed in vacuo. The

crude product was diluted with 100 vol of distilled water and

extracted with chloroform (3x). The organic layer was collected,

washed with brine, and dried with MgSO₄before removing the

volatiles in vacuo.

2-Bromo-N-phenylacetamide (11a). 2-Bromoacetyl bromide 10

(200 mg, 86 μL 0.991 mmol, 1 equiv) was reacted with aniline (76.9

mg, 78 μL, 0.825 mmol, 0.83 equiv) according to the general

procedure method A. The residue was then puriﬁed over silica gel

chromatography using 5−40% ethyl acetate in petroleum spirit to

yield the desired product as a white powder (210 mg, 99%): LC-MS:

1

R_t= 2.48 min, 99 Abs % @ 200 nm, [M + H]⁺= 216.1; H NMR

Synthetic Procedures. General Procedure for the Preparation

of 2-Bromoacetamides. Method A: Bromoacetyl bromide (1.0

equiv) was added dropwise to a stirring solution of amine (0.83

equiv) and triethylamine (1.0 equiv) in 20 vol of anhydrous

dichloromethane at 0 °C under nitrogen. The reaction mixture was

stirred at rt for 1−24 h and then concentrated under reduced pressure

after completion of reaction.

General Procedure for Alkylation of Imidazopyrazinone. Method

B: To a stirring suspension of imidazopyrazinone (1 equiv) in dry

DMF (20 vol) was added 3 equiv of cesium carbonate or potassium

carbonate, followed by addition of 1.2−1.5 equiv of benzyl bromide in

dropwise. The reaction was either stirred at room temperature or

microwaved at 80−100 °C.

(600 MHz, DMSO-d₆) δ 10.39 (s, 1H), 7.61−7.56 (m, 2H), 7.36−

7.29 (m, 2H), 7.08 (tt, J = 7.4, 1.2 Hz, 1H), 4.04 (s, 2H).

N-Benzyl-2-bromoacetamide (11b). 2-Bromoacetyl bromide 10

(200 mg, 86 μL 0.991 mmol, 1 equiv) was reacted with benzylamine

(88.5 mg, 90 μL, 0.825 mmol, 0.83 equiv) according to the general

procedure method A. The residue was then puriﬁed over silica gel

chromatography using 5−50% ethyl acetate in petroleum spirit to

yield the desired product as a white powder (177 mg, 79%): LC-MS:

1

R_t= 2.48 min, 99 Abs % @ 200 nm, [M + H]⁺= 228.1; H NMR

(600 MHz, DMSO-d₆) δ 8.77 (t, J = 6.1 Hz, 1H), 7.37−7.31 (m, 2H),

7.30−7.23 (m, 2H), 4.29 (d, J = 5.9 Hz, 2H), 3.91 (s, 2H).

2-Bromo-N-(3-(triﬂuoromethoxy)benzyl)acetamide (11c). 2-Bro-

moacetyl bromide 10 (200 mg, 118 μL 0.991 mmol, 1 equiv) was

reacted with 3-triﬂuoromethoxy benzylamine (157 mg, 129 μL, 0.825

mmol, 0.83 equiv) for 1 h according to the general procedure method

A. The residue was then puriﬁed over silica gel chromatography using

0−4% methanol in dichloromethane to yield the desired product as a

Method C: To a stirring suspension of imidazopyrazinone (1

equiv) in dry DMF (20 vol) at 0 °C was added 1.5 equiv of 60%

sodium hydride followed by addition of 1.2−1.5 equiv of benzyl

bromide dropwise. The reaction was stirred at room temperature

overnight under N₂.

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white powder (181 mg, 59%). LC-MS: R_t= 2.91 min, 99 Abs % @

200 nm, [M + H]⁺= 312.0; ¹H NMR (600 MHz, DMSO-d₆) δ 8.89−

8.84 (m, 1H), 7.50−7.44 (m, 1H), 7.32−7.28 (m, 1H), 7.27−7.22

(m, 1H), 4.35 (d, J = 6.0 Hz, 2H), 3.92 (s, 2H).

1H), 4.80 (s, 2H). ¹³C NMR (150 MHz, DMSO) δ 165.0, 152.8,

148.0, 138.5, 134.8, 128.9, 124.8, 123.6, 119.1, 116.7, 106.6, 50.6.

HRMS (ESI): m/z calcd for C₁₄H₁₂N₅O₄[M + H]⁺, 314.0884; found,

314.0892.

2-Nitro-7-(2-oxo-2-phenylethyl)imidazo[1,2-a]pyrazin-8(7H)-

one (17a). 2-Nitroimidazo[1,2-a]pyrazin-8(7H)-one 16 (50 mg,

0.278 mmol, 1 equiv) was reacted with phenacyl bromide (82.9 mg,

0.416 mmol, 1.5 equiv) at room temperature for 30 min according to

the general procedure method B. After the reaction was completed as

analyzed by LC-MS, the mixture was diluted with distilled water (15

mL) and the precipitate was collected by ﬁltration. The precipitate

was then puriﬁed by recrystallization using hot ACN/MeOH to yield

the desired product, 2-nitro-7-(2-oxo-2-phenylethyl)imidazo[1,2-a]-

pyrazin-8(7H)-one 17a, as a brown solid (30 mg, 36%): LC-MS: R_t=

N-Benzyl-2-(2-nitro-8-oxoimidazo[1,2-a]pyrazin-7(8H)-yl)-

acetamide (17e). 2-Nitroimidazo[1,2-a]pyrazin-8(7H)-one 16 (70

mg, 0.389 mmol, 1 equiv) was reacted with N-benzyl-2-bromoace-

tamide 11b (106 mg, 0.466 mmol, 1.2 equiv) under microwave

conditions at 90 °C for 15 min according to the general procedure

method B. After the reaction was completed as analyzed by LC-MS,

the mixture was diluted with distilled water (20 mL) and the

precipitate was collected by ﬁltration. The precipitate was then

puriﬁed by recrystallization using hot ACN to yield the desired

product, N-benzyl-2-(2-nitro-8-oxoimidazo[1,2-a]pyrazin-7(8H)-yl)-

acetamide 17e, as a white solid (93 mg, 73%): LC-MS: R_t= 2.46

1

2.60 min, 99 Abs % @ 254 nm, [M + H]⁺= 299.1; H NMR (600

1

min, 99 Abs % @ 254 nm, [M + H]⁺= 328.1; H NMR (600 MHz,

MHz, DMSO-d₆) δ 8.89 (s, 1H), 8.12−8.06 (m, 2H), 7.75 (ddt, J =

7.8, 7.1, 1.3 Hz, 1H), 7.66 (d, J = 5.8 Hz, 1H), 7.65−7.59 (m, 2H),

7.34 (d, J = 5.8 Hz, 1H), 5.60 (s, 2H). ¹³C NMR (150 MHz, DMSO)

δ 192.3, 152.7, 148.1, 134.7, 134.2, 134.2, 129.0, 128.1, 124.4, 116.9,

107.0, 54.2. HRMS (ESI): m/z calcd for C₁₄H₁₁N₄O₄[M + H]⁺,

299.0775; found, 299.0785.

DMSO-d₆) δ 8.84 (s, 1H), 8.69 (t, J = 6.0 Hz, 1H), 7.59 (d, J = 5.9

Hz, 1H), 7.37−7.30 (m, 3H), 7.30−7.22 (m, 3H), 4.64 (s, 2H), 4.32

(d, J = 6.0 Hz, 2H). ¹³C NMR (150 MHz, DMSO) δ 166.3, 152.9,

147.9, 138.9, 135.0, 128.3, 127.1, 126.8, 124.7, 116.6, 106.7, 50.2,

42.1. HRMS (ESI): m/z calcd for C₁₅H₁₄N₅O₄[M + H]⁺, 328.1040;

found, 328.1054.

2-Nitro-7-(2-oxo-2-(4-(triﬂuoromethoxy)phenyl)ethyl)imidazo-

[1,2-a]pyrazin-8(7H)-one (17b). 2-Nitroimidazo[1,2-a]pyrazin-

8(7H)-one 16 (42 mg, 0.233 mmol, 1 equiv) was reacted with 2-

bromo-1-(4-(triﬂuoromethoxy)phenyl)ethan-1-one (99 mg, 0.350

mmol, 1.5 equiv) at room temperature for 30 min according to the

general procedure method B. After the reaction was completed as

analyzed by LC-MS, the mixture was diluted with distilled water (15

mL) and the precipitate was collected by ﬁltration. The precipitate

was then puriﬁed by recrystallization using hot ACN/MeOH to yield

the desired product, 2-nitro-7-(2-oxo-2-phenylethyl)imidazo[1,2-a]-

pyrazin-8(7H)-one 17b, as a yellow solid (37 mg, 41%): LC-MS: R_t=

2-(2-Nitro-8-oxoimidazo[1,2-a]pyrazin-7(8H)-yl)-N-(3-

(triﬂuoromethoxy)benzyl)acetamide (17f). 2-Nitroimidazo[1,2-a]-

pyrazin-8(7H)-one 16 (50 mg, 0.278 mmol, 1 equiv) was reacted with

2-bromo-N-(3-(triﬂuoromethoxy)benzyl)acetamide 11c (130 mg,

0.416 mmol, 1.5 equiv) under microwave conditions at 90 °C for

15 min according to the general procedure method B. After the

reaction was completed as analyzed by LC-MS, the mixture was

diluted with distilled water (15 mL) and the precipitate was collected

by ﬁltration. The precipitate was then puriﬁed by recrystallization

using hot ACN to yield the desired product, 2-(2-nitro-8-oxoimidazo-

[1,2-a]pyrazin-7(8H)-yl)-N-(3-(triﬂuoromethoxy)benzyl)acetamide

17f, as a white solid (25 mg, 22%): LC-MS: R_t= 2.79 min, 99 Abs %

1

2.91 min, 99 Abs % @ 254 nm, [M + H]⁺= 383.1; H NMR (600

MHz, DMSO-d₆) δ 8.89 (s, 1H), 8.26−8.20 (m, 2H), 7.66 (d, J = 5.8

Hz, 1H), 7.64−7.58 (m, 2H), 7.32 (d, J = 5.8 Hz, 1H), 5.61 (s, 2H).

¹³C NMR (150 MHz, DMSO) δ 191.3, 152.7, 152.1, 148.1, 134.7,

133.1, 130.7, 124.3, 121.1, 119.9 (q, J = 258.3 Hz), 116.9, 107.0, 54.2.

HRMS (ESI): m/z calcd for C₁₅H₁₀F₃N₄O₅[M + H]⁺, 383.0598;

found, 383.0605.

1

@ 254 nm, [M + H]⁺= 412.1; H NMR (600 MHz, DMSO-d₆) δ

8.84 (s, 1H), 8.76 (t, J = 6.0 Hz, 1H), 7.60 (d, J = 5.9 Hz, 1H), 7.47

(t, J = 7.8 Hz, 1H), 7.37−7.29 (m, 2H), 7.27−7.22 (m, 2H), 4.65 (s,

2H), 4.37 (d, J = 6.0 Hz, 2H). ¹³C NMR (150 MHz, DMSO) δ 166.6,

152.0, 148.4, 147.9, 142.0, 135.0, 130.2, 126.1, 124.7, 120.1 (q, J =

256.7 Hz), 119.5, 119.3, 116.6, 106.7, 50.2, 41.6. HRMS (ESI): m/z

calcd for C₁₆H₁₃F₃N₅O₅[M + H]⁺, 412.0863; found, 412.0856.

2-(2-Nitro-8-oxoimidazo[1,2-a]pyrazin-7(8H)-yl)ethyl Acetate

(17g). 2-Nitroimidazo[1,2-a]pyrazin-8(7H)-one 16 (130 mg, 0.722

mmol, 1 equiv) was reacted with 2-bromoethyl acetate (181 mg, 1.08

mmol, 1.5 equiv) under microwave conditions at 80 °C for 30 min

according to the general procedure method B. After the reaction was

completed as analyzed by LC-MS, the mixture was diluted with

distilled water (15 mL) and the precipitate was collected by ﬁltration

to yield the desired product, 2-(2-nitro-8-oxoimidazo[1,2-a]pyrazin-

7(8H)-yl)ethyl acetate 17g, as a cream solid (171 mg, 89%): LC-MS:

2-Nitro-7-(2-oxo-2-(p-tolyl)ethyl)imidazo[1,2-a]pyrazin-8(7H)-

one (17c). 2-Nitroimidazo[1,2-a]pyrazin-8(7H)-one 16 (50 mg,

0.278 mmol, 1 equiv) was reacted with 2-bromo-4′-methylacetophe-

none (88.7 mg, 0.416 mmol, 1.5 equiv) at room temperature for 2 h

according to the general procedure method B. After the reaction was

completed as analyzed by LC-MS, the mixture was diluted with

distilled water (15 mL) and the precipitate was collected by ﬁltration.

The precipitate was then puriﬁed by recrystallization using hot ACN/

MeOH to yield the desired product, 2-nitro-7-(2-oxo-2-(p-tolyl)-

ethyl)imidazo[1,2-a]pyrazin-8(7H)-one 17c, as a red solid (20 mg,

23%): LC-MS: R_t= 2.74 min, 98 Abs % @ 254 nm, [M + H]⁺=

1

R_t= 2.14 min, 99 Abs % @ 254 nm, [M + H]⁺= 267.1; H NMR

1

313.1; H NMR (600 MHz, DMSO-d₆) δ 8.88 (s, 1H), 8.01−7.96

(600 MHz, DMSO-d₆) δ 8.81 (s, 1H), 7.57 (d, J = 5.8 Hz, 1H), 7.35

(d, J = 5.8 Hz, 1H), 4.30 (t, J = 5.2 Hz, 2H), 4.16 (t, J = 5.2 Hz, 2H),

1.97 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 170.2, 152.8, 147.9,

134.9, 124.0, 116.5, 106.8, 61.3, 46.7, 20.5. HRMS (ESI): m/z calcd

for C₁₀H₁₀N₄NaO₅[M + Na]⁺, 289.0543; found, 289.0545.

(m, 2H), 7.65 (d, J = 5.8 Hz, 1H), 7.45−7.40 (m, 2H), 7.33 (d, J =

5.9 Hz, 1H), 5.56 (s, 2H), 2.43 (s, 3H); ¹³C NMR (150 MHz,

DMSO) δ 191.8, 152.7, 148.1, 144.8, 134.7, 131.7, 129.5, 128.2,

124.4, 116.8, 106.9, 54.1, 21.3. HRMS (ESI): m/z calcd for

C₁₅H₁₃N₄O₄[M + H]⁺, 313.0931; found, 313.0941.

7-(2-Hydroxyethyl)-2-nitroimidazo[1,2-a]pyrazin-8(7H)-one

(17h). To a stirring suspension of 2-(2-nitro-8-oxoimidazo[1,2-

a]pyrazin-7(8H)-yl)ethyl acetate 17g (165 mg, 0.266 mmol, 1

equiv) in methanol (20 vol) was added 1.5 equiv of potassium

carbonate (128 mg, 0.930 mmol). The reaction was stirred for 1 h at

room temperature before removing the volatiles under N₂. The solid

was suspended with 5% TFA in methanol and puriﬁed by C18-

reversed phase silica (Grace Reveleris X2, A: 0.1% TFA in water, B:

0.1% TFA in ACN, 0−50% B) to give ﬁnal product, 7-(2-

hydroxyethyl)-2-nitroimidazo[1,2-a]pyrazin-8(7H)-one 17h, as a

light yellow solid (47 mg, 34%): LC-MS: R_t= 1.53 min, 99 Abs %

2-(2-Nitro-8-oxoimidazo[1,2-a]pyrazin-7(8H)-yl)-N-phenylaceta-

mide (17d). 2-Nitroimidazo[1,2-a]pyrazin-8(7H)-one 16 (75 mg,

0.416 mmol, 1 equiv) was reacted with 2-bromo-N-phenylacetamide

11a (107 mg, 0.500 mmol, 1.2 equiv) under microwave conditions at

90 °C for 15 min according to the general procedure method B. After

the reaction was completed as analyzed by LC-MS, the mixture was

diluted with distilled water (20 mL) and the precipitate was collected

by ﬁltration. The precipitate was then puriﬁed by recrystallization

using hot ACN/MeOH to yield the desired product, 2-(2-nitro-8-

oxoimidazo[1,2-a]pyrazin-7(8H)-yl)-N-phenylacetamide 17d, as a

white solid (21 mg, 16%): LC-MS: R_t= 2.46 min, 99 Abs % @

254 nm, [M + H]⁺= 314.1; ¹H NMR (600 MHz, DMSO-d₆) δ 10.34

(s, 1H), 8.86 (s, 1H), 7.63 (d, J = 5.9 Hz, 1H), 7.60−7.55 (m, 2H),

7.38 (d, J = 5.8 Hz, 1H), 7.36−7.29 (m, 2H), 7.07 (tt, J = 7.3, 1.2 Hz,

1

@ 254 nm, [M + H]⁺= 225.1; H NMR (600 MHz, DMSO-d₆) δ

8.81 (s, 1H), 7.55 (d, J = 5.9 Hz, 1H), 7.29 (d, J = 5.8 Hz, 1H), 4.90

(t, J = 5.7 Hz, 1H), 3.96 (t, J = 5.5 Hz, 2H), 3.66 (q, J = 5.5 Hz, 2H).

M

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¹³C NMR (150 MHz, DMSO-d₆) δ 152.8, 147.9, 135.1, 124.8, 116.3,

2-(Chloromethyl)-1-methyl-1H-imidazole (19). (1-Methyl-1H-

imidazol-2-yl)methanol (300 mg, 2.68 mmol, 1 equiv) and thionyl

chloride (388 μL, 5.35 mmol, 2 equiv) were reacted and puriﬁed

according to the general procedure method D. Volatiles were then

removed in vacuo a crude compound was then recrystallized from

ethanol to give 2-(chloromethyl)-1-methyl-1H-imidazole 19 (220 mg,

63%) as a colorless crystal: LC-MS (Waters Atlantis T3): R_t= 0.29

106.3, 58.5, 50.3. HRMS (ESI): m/z calcd for C₈H₈N₄NaO₄[M +

Na]⁺, 247.0438; found, 247.0435.

7-(2-(Benzyloxy)ethyl)-2-nitroimidazo[1,2-a]pyrazin-8(7H)-one

(17i). 7-(2-Hydroxyethyl)-2-nitroimidazo[1,2-a]pyrazin-8(7H)-one

17h (20 mg, 0.0892 mmol, 1 equiv) was reacted with benzyl bromide

(18.3 mg, 12.7 μL, 0.107 mmol, 1.2 equiv) according to the general

procedure method C. Additional 1.2 equiv of benzyl bromide and 1.5

equiv of sodium hydride were added to push the reaction. Once the

reaction was completed as analyzed by LC-MS, the mixture was

diluted with distilled water and extracted with EtOAc three times. The

crude was then puriﬁed by C18-reversed phase silica (Grace Reveleris

X2, A: 0.1% TFA in water, B: 0.1% TFA in ACN, 0−100% B) to give

ﬁnal product, 7-(2-(benzyloxy)ethyl)-2-nitroimidazo[1,2-a]pyrazin-

8(7H)-one 17i, as a white solid (8.1 mg, 29%): LC-MS: R_t= 2.76

1

min, 95 Abs % @ 210 nm, [M + H]⁺= 131.0. H NMR (600 MHz,

DMSO-d₆) δ 7.76 (d, J = 1.9 Hz, 1H), 7.69 (d, J = 1.9 Hz, 1H), 5.18

(s, 2H), 3.87 (s, 3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 141.5,

124.6, 119.4, 34.2, 31.7.

5-(Chloromethyl)-2-(triﬂuoromethyl)pyridine (20). (6-

(Triﬂuoromethyl)pyridin-3-yl)methanol (200 mg, 1.13 mmol, 1

equiv) and phosphoryl chloride (571 μL, 1.65 mmol, 3.3 equiv)

were reacted and puriﬁed according to the general procedure method

D. Volatiles were removed in vacuo to give 5-(chloromethyl)-2-

(triﬂuoromethyl)pyridine 20 in a gel form, which was used in the next

step immediately without further puriﬁcation: LC-MS: R_t= 2.59 min,

96 Abs % @ 254 nm, [M + H]⁺= 196.1. This halide intermediate is

unstable, and therefore NMR characterization data was not acquired.

5-(Bromomethyl)-1-methyl-1H-pyrazole (21). (1-Methyl-1H-pyr-

azol-5-yl)methanol (195 mg, 1.74 mmol, 1 equiv) was added with

DCM (80 vol) and was chilled at 0 °C. Phosphorus tribromides (343

μL, 3.65 mmol, 2.1 equiv) was added dropwise, and the reaction was

continued to stir at room temperature for 6 h. The mixture was then

diluted with distilled water, adjusted to pH 8−9 using saturated

NaHCO₃, and extracted with DCM (3 x 40 mL). The organic layer

was collected and washed with brine, and volatiles were removed in

vacuo to give 5-(bromomethyl)-1-methyl-1H-pyrazole 21 (272 mg,

89%), which was used in the next step immediately without further

puriﬁcation: LC-MS: R_t= 2.28 min, 99 Abs % @ 254 nm, [M + H]⁺=

177.1. This halide intermediate is unstable, and therefore NMR

characterization data was not acquired.

2-(Chloromethyl)-1H-benzo[d]imidazole (24). To a stirring

solution of o-phenylenediamine 22 (500 mg, 4.62 mmol, 1 equiv)

in 4 M HCl (12 vol) was added 1.2 equiv of chloroacetic acid (524

mg, 5.55 mmol, 1.2 equiv). The mixture was reﬂux overnight and then

cooled to room temperature before neutralization with saturated

NaHCO₃. Precipitate formed was ﬁltered under vacuum and washed

with cold water to yield 2-(chloromethyl)-1H-benzo[d]imidazole 24

as a yellow solid (633 mg, 82%): LC-MS: R_t= 1.65 min, 98 Abs % @

254 nm, [M + H]⁺= 167.2; ¹H NMR (600 MHz, DMSO-d₆) δ 7.58−

7.51 (m, 2H), 7.21 (dp, J = 7.1, 4.0 Hz, 2H), 4.92 (s, 2H).¹³C NMR

(150 MHz, DMSO) δ 149.6, 122.3, 38.4.

1

min, 99 A % @ 254 nm, [M + H]⁺= 315.1; H NMR (600 MHz,

DMSO-d₆) δ 8.81 (s, 1H), 7.56 (d, J = 5.9 Hz, 1H), 7.33 (d, J = 5.9

Hz, 1H), 7.31−7.20 (m, 5H), 4.49 (s, 2H), 4.13 (t, J = 5.3 Hz, 2H),

3.71 (t, J = 5.3 Hz, 2H). ¹³C NMR (151 MHz, DMSO) δ 152.7,

147.9, 138.0, 134.9, 128.1, 127.4, 124.4, 116.4, 106.4, 71.8, 67.0, 47.4.

HRMS (ESI): m/z calcd for C₁₅H₁₅N₄O₄[M + H]⁺, 315.1088; found,

315.1100.

2-Nitro-7-(2-((4-(triﬂuoromethoxy)benzyl)oxy)ethyl)imidazo[1,2-

a]pyrazin-8(7H)-one (17j). 7-(2-Hydroxyethyl)-2-nitroimidazo[1,2-

a]pyrazin-8(7H)-one 17 h (57 mg, 0.254 mmol, 1 equiv) was reacted

with 1-(bromomethyl)-4-(triﬂuoromethoxy)benzene (77.8 mg, 48.8

μL, 0.305 mmol, 1.2 equiv) according to the general procedure

method C. After completion of reaction, the mixture was diluted with

distilled water (12 mL) and extracted with EtOAc (3 × 12 mL). The

organic layer was collected, washed with brine (12 mL), and dried

with MgSO₄. Volatiles were removed in vacuo, and the crude product

was puriﬁed over silica gel by MPLC (Biotage Isolera, 0−8% DCM/

MeOH), then re-puriﬁed by C18-reversed phase silica (Grace

Reveleris X2, A: 0.1% TFA in water, B: 0.1% TFA in ACN, 0−

100% B) to give ﬁnal product, 2-nitro-7-(2-((4-(triﬂuoromethoxy)-

benzyl)oxy)ethyl)imidazo[1,2-a]pyrazin-8(7H)-one 17j, as a beige

solid (16 mg, 16%): LC-MS: R_t= 3.00 min, 99 Abs % @ 254 nm, [M

+ H]⁺= 399.1; ¹H NMR (600 MHz, DMSO-d₆) δ 8.80 (s, 1H), 7.56

(d, J = 5.9 Hz, 1H), 7.42−7.36 (m, 2H), 7.33 (d, J = 5.9 Hz, 1H),

7.29−7.24 (m, 2H), 4.51 (s, 2H), 4.13 (t, J = 5.2 Hz, 2H), 3.73 (t, J =

5.2 Hz, 2H). ¹³C NMR (150 MHz, DMSO) δ 152.7, 147.9, 147.5,

137.6, 134.9, 129.2, 124.4, 120.7, 120.1 (q, J = 256 Hz), 116.4, 106.5,

70.8, 67.2, 47.4. HRMS (ESI): m/z calcd for C₁₆H₁₄F₃N₄O₅[M +

H]⁺, 399.0911; found, 399.0917.

2-(Chloromethyl)-1-methyl-1H-benzo[d]imidazole (25). To a

stirring solution of N-methyl-1,2-phenylenediamine 23 (500 mg,

4.09 mmol, 1 equiv) in 4 M HCl (12 vol) was added 1.2 equiv of

chloroacetic acid (580 mg, 6.14 mmol, 1.5 equiv). The mixture was

reﬂux overnight and then cooled to room temperature before

neutralization with saturated NaHCO₃. Precipitate formed was

ﬁltered under vacuum and washed with cold water to yield 2-

(chloromethyl)-1-methyl-1H-benzo[d]imidazole 25 as a black solid

(524 mg, 71%): LC-MS: R_t= 2.15 min, 99 Abs % @ 254 nm, [M +

H]⁺= 181.1; ¹H NMR (600 MHz, DMSO-d₆) δ 7.63 (dt, J = 8.0, 0.9

Hz, 1H), 7.58 (dt, J = 8.1, 1.0 Hz, 1H), 7.30 (ddd, J = 8.2, 7.1, 1.1 Hz,

1H), 7.23 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 5.08 (s, 2H), 3.86 (s, 3H).

¹³C NMR (150 MHz, DMSO) δ 149.6, 141.7, 135.9, 123.0, 122.0,

7-(2-((4-Methylbenzyl)oxy)ethyl)-2-nitroimidazo[1,2-a]pyrazin-

8(7H)-one (17k). 7-(2-Hydroxyethyl)-2-nitroimidazo[1,2-a]pyrazin-

8(7H)-one 17h (55 mg, 0.245 mmol, 1 equiv) was reacted with 4-

methylbenzyl bromide (54.5 mg, 34.2 μL, 0.294 mmol, 1.2 equiv)

according to the general procedure method C. After completion of

reaction, the mixture was diluted with distilled water (11 mL) and the

precipitate was collected by ﬁltration. The precipitate was then

puriﬁed by recrystallization (slurry in hot ACN) to yield the desired

product, 7-(2-((4-methylbenzyl)oxy)ethyl)-2-nitroimidazo[1,2-a]-

pyrazin-8(7H)-one 17k, as a beige solid (62 mg, 76%): LC-MS: R_t

= 2.83 min, 99 Abs % @ 254 nm, [M + H]⁺= 329.1; ¹H NMR (600

MHz, DMSO-d₆) δ 8.80 (d, J = 1.0 Hz, 1H), 7.55 (dd, J = 5.8, 0.9 Hz,

1H), 7.30 (dd, J = 5.9, 0.9 Hz, 1H), 7.12 (d, J = 7.7 Hz, 2H), 7.05 (d,

J = 7.7 Hz, 2H), 4.42 (s, 2H), 4.09 (t, J = 5.2 Hz, 2H), 3.68 (t, J = 5.2

Hz, 2H), 2.22 (s, 3H).¹³C NMR (150 MHz, DMSO) δ 152.6, 147.9,

136.6, 134.9, 134.9, 128.6, 127.6, 124.5, 116.4, 106.4, 71.7, 66.7, 47.5,

20.6. HRMS (ESI): m/z calcd for C₁₆H₁₇N₄O₄[M + H]⁺, 329.1244;

found, 329.1258.

5-(Chloromethyl)thiazole (18). Thiazol-5-ylmethanol (300 mg,

2.61 mmol, 1 equiv) and thionyl chloride (567 μL, 7.82 mmol, 3

equiv) were reacted and puriﬁed according to the general procedure

method D. Volatiles were removed in vacuo to give 5-(chloromethyl)-

thiazole 18 (264 mg, 76%), which was used in the next step

immediately: LC-MS: R_t= 2.23 min, 99 Abs % @ 254 nm, [M + H]⁺

= 134.1. This halide intermediate is unstable, and therefore NMR

characterization data was not acquired.

119.3, 110.4, 37.1, 30.0.

2-Nitro-7-(thiazol-5-ylmethyl)imidazo[1,2-a]pyrazin-8(7H)-one

(26a). 2-Nitroimidazo[1,2-a]pyrazin-8(7H)-one 16 (55 mg, 0.305

mmol, 1 equiv) was reacted with 5-(chloromethyl)thiazole 18 (61.2

mg, 0.458 mmol, 1.5 equiv) under microwave conditions at 90 °C (20

min x 2) according to the general procedure method B. After the

reaction was completed as analyzed by LC-MS, the mixture was

diluted with distilled water (15 mL) and the precipitate was collected

by ﬁltration. The precipitate was then puriﬁed by recrystallization

using DCM/MeOH (hot slurry) to yield the desired product, 2-nitro-

7-(thiazol-5-ylmethyl)imidazo[1,2-a]pyrazin-8(7H)-one, 26a as a

cream solid (54 mg, 62%): LC-MS: R_t= 2.23 min, 99 Abs % @

1

254 nm, [M + H]⁺= 278.1; H NMR (600 MHz, DMSO-d₆) δ 9.05

N

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(d, J = 0.8 Hz, 1H), 8.81 (s, 1H), 8.01 (d, J = 0.8 Hz, 1H), 7.61 (d, J =

5.9 Hz, 1H), 7.50 (d, J = 5.9 Hz, 1H), 5.36 (s, 2H); ¹³C NMR (150

MHz, DMSO-d₆) δ 155.6, 152.5, 148.0, 143.3, 134.8, 132.8, 122.8,

116.8, 107.8, 42.8. HRMS (ESI): m/z calcd for C₁₀H₈N₅O₃S [M +

H]⁺, 278.0342; found, 278.0348.

distilled water (20 mL) and the precipitate was collected by ﬁltration.

The precipitate was then puriﬁed by recrystallization using DCM/

MeOH (hot slurry) to yield the desired product, 7-((1-methyl-1H-

benzo[d]imidazol-2-yl)methyl)-2-nitroimidazo[1,2-a]pyrazin-8(7H)-

one 26f, as a light gray solid (135 mg, 75%): LC-MS: R_t= 2.30 min,

1

7-((1-Methyl-1H-imidazol-2-yl)methyl)-2-nitroimidazo[1,2-a]-

pyrazin-8(7H)-one (26c). 2-Nitroimidazo[1,2-a]pyrazin-8(7H)-one

16 (50 mg, 0.278 mmol, 1 equiv) was reacted with 2-

(chloromethyl)-1-methyl-1H-imidazole 19 (54.4 mg, 0.416 mmol,

1.5 equiv) under microwave conditions at 90 °C for 20 min according

to the general procedure method B. After the reaction was completed

as analyzed by LC-MS, the mixture was diluted with distilled water

(15 mL) and the precipitate was collected by ﬁltration. The

precipitate was then puriﬁed by recrystallization using DCM/

MeOH (hot slurry) to yield the desired product, 7-((1-methyl-1H-

imidazol-2-yl)methyl)-2-nitroimidazo[1,2-a]pyrazin-8(7H)-one 26c,

as a white solid (60 mg, 79%): LC-MS: R_t= 1.07 min, 99 Abs %

99 Abs % @ 254 nm, [M + H]⁺= 325.1; H NMR (600 MHz,

DMSO-d₆) δ 8.87 (s, 1H), 7.65 (d, J = 5.9 Hz, 1H), 7.59−7.52 (m,

2H), 7.44 (d, J = 5.9 Hz, 1H), 7.25 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H),

7.17 (ddd, J = 8.1, 7.1, 1.2 Hz, 1H), 5.47 (s, 2H), 3.88 (s, 3H. ¹³C

NMR (150 MHz, DMSO-d₆) δ 152.6, 149.7, 148.0, 141.8, 136.0,

134.8, 124.1, 122.3, 121.6, 118.8, 116.8, 110.1, 107.3, 43.4, 29.8.

HRMS (ESI): m/z calcd for C₁₅H₁₃N₆O₃[M + H]⁺, 325.1044; found,

325.1061.

2-Nitro-7-((6-(triﬂuoromethyl)pyridin-3-yl)methyl)imidazo[1,2-

a]pyrazin-8(7H)-one (26h). 2-Nitroimidazo[1,2-a]pyrazin-8(7H)-

one 16 (50 mg, 0.278 mmol, 1 equiv) was reacted with 3-(4-

(chloromethyl)-2-(triﬂuoromethyl)pyridine 20 (65 mg, 0.333 mmol,

1.2 equiv) under microwave conditions at 100 °C for 30 min

according to the general procedure method B. After the reaction was

completed as analyzed by LC-MS, the mixture was diluted with

distilled water (20 mL) and the precipitate was collected by ﬁltration.

The precipitate was then puriﬁed by recrystallization using DCM/

MeOH (hot slurry) to yield the desired product, 2-nitro-7-((6-

(triﬂuoromethyl)pyridin-3-yl)methyl)imidazo[1,2-a]pyrazin-8(7H)-

one 26h, as a white solid (45 mg, 48% yield). Filtrate from DCM/

MeOH was collected and further puriﬁed over C18-reversed phase

silica (Grace Reveleris X2, A: 0.1% TFA in water, B: 0.1% TFA in

ACN, 5−100% B) to give additional product 26h (14 mg, 15%): LC-

MS: R_t= 2.43 min, 99 Abs % @ 254 nm, [M + H]⁺= 340.1; ¹H NMR

(600 MHz, DMSO-d₆) δ 8.83 (s, 1H), 8.81 (d, J = 2.1 Hz, 1H),

8.07−8.02 (m, 1H), 7.90 (dd, J = 8.2, 0.8 Hz, 1H), 7.63 (d, J = 5.9

Hz, 1H), 7.51 (d, J = 5.9 Hz, 1H), 5.28 (s, 2H). ¹³C NMR (151 MHz,

DMSO) δ 153.0, 149.6, 147.9, 145.6 (J = 33.9 Hz), 137.5, 136.2,

135.2, 123.4, 122.5, 121.6 (J = 273.3 Hz), 120.6 (J = 2.8 Hz), 107.7,

48.0. HRMS (ESI): m/z calcd for C₁₃H₉F₃N₅O₃[M + H]⁺, 340.0652;

found, 340.0665.

4-Nitro-N-(thiazol-2-ylmethyl)-1H-imidazole-2-carboxamide

(27b). A mixture of acid chloride crude solid (150 mg, 0.855 mmol, 1

equiv) and triethylamine (238 μL, 1.71 mmol, 2 equiv) was reacted

with thiazol-2-ylmethanamine (117 mg, 97 μL, 1.03 mmol, 1.2 equiv)

according to general procedure method E. After completion of

reaction, volatiles were removed in vacuo before purifying over C18-

reversed phase silica (Grace Reveleris X2, A: 0.1% TFA in water, B:

0.1% TFA in ACN, 0−100% B) to give the ﬁnal product, 4-nitro-N-

(thiazol-2-ylmethyl)-1H-imidazole-2-carboxamide 27b, as a white

solid (166 mg, 77%). LC-MS: R_t= 2.15 min, 99 Abs % @ 254 nm,

[M + H]⁺= 254.1; ¹H NMR (600 MHz, DMSO-d₆) δ 9.74 (t, J = 6.2

Hz, 1H), 8.49 (s, 1H), 7.73 (d, J = 3.2 Hz, 1H), 7.63 (d, J = 3.3 Hz,

1H), 4.72 (d, J = 6.1 Hz, 2H). ¹³C NMR (150 MHz, DMSO) δ 168.4,

157.5, 146.8, 142.2, 139.1, 121.7, 120.1, 40.6.

4-Nitro-N-(pyridin-3-ylmethyl)-1H-imidazole-2-carboxamide

(27g). A mixture of acid chloride crude solid (120 mg, 0.684 mmol, 1

equiv) and triethylamine (191 μL, 1.37 mmol, 2 equiv) was reacted

with pyridin-3-ylmethanamine (88.8 mg, 0.820 mmol, 1.2 equiv)

according to general procedure method E. After completion of

reaction, volatiles were removed in vacuo before purifying over C18-

reversed phase silica (Grace Reveleris X2, A: 0.1% TFA in water, B:

0.1% TFA in ACN, 0−50% B) to give the ﬁnal product, 4-nitro-N-

(pyridin-3-ylmethyl)-1H-imidazole-2-carboxamide 27g, as a white

solid (128 mg, 76%). LC-MS: R_t= 1.21 min, 99% Abs @ 254 nm, [M

+ H]⁺= 248.1; ¹H NMR (600 MHz, DMSO-d₆) δ 9.57 (t, J = 6.2 Hz,

1H), 8.73 (s, 1H), 8.65 (d, J = 4.8 Hz, 1H), 8.48 (s, 1H), 8.13 (dt, J =

8.1, 1.8 Hz, 1H), 7.70 (dd, J = 7.9, 5.2 Hz, 1H), 4.55 (d, J = 6.2 Hz,

2H). Triethylamine was presented as 5% when integrated by ¹H

NMR.

1

@ 254 nm, [M + H]⁺= 275.1; H NMR (600 MHz, DMSO-d₆) δ

8.83 (s, 1H), 7.59 (d, J = 5.9 Hz, 1H), 7.31 (d, J = 5.9 Hz, 1H), 7.14

(d, J = 1.2 Hz, 1H), 6.81 (d, J = 1.2 Hz, 1H), 5.18 (s, 2H), 3.69 (s,

3H). ¹³C NMR (150 MHz, DMSO) δ 152.5, 148.0, 142.2, 134.9,

127.0, 123.4, 122.4, 116.7, 107.3, 42.1, 32.5. HRMS (ESI): m/z calcd

for C₁₁H₁₁N₆O₃[M + H]⁺, 275.0887; found, 275.0893.

7-((1-Methyl-1H-pyrazol-5-yl)methyl)-2-nitroimidazo[1,2-a]-

pyrazin-8(7H)-one (26d). 2-Nitroimidazo[1,2-a]pyrazin-8(7H)-one

16 (60 mg, 0.333 mmol, 1 equiv) was reacted with 5-(bromomethyl)-

1-methyl-1H-pyrazole 21 (87.5 mg, 0.500 mmol, 1.5 equiv) under

microwave conditions at 90 °C for 20 min according to the general

procedure method B. After the reaction was completed as analyzed by

LC-MS, the mixture was diluted with distilled water (15 mL) and the

precipitate was collected by ﬁltration. The precipitate was then

puriﬁed by recrystallization using DCM/MeOH (hot slurry) to yield

the desired product, 7-((1-methyl-1H-pyrazol-5-yl)methyl)-2-

nitroimidazo[1,2-a]pyrazin-8(7H)-one 26d, as a cream color solid

(57 mg, 63%): LC-MS: R_t= 2.20 min, 98 Abs % @ 254 nm, [M +

H]⁺= 275.2; ¹H NMR (600 MHz, DMSO-d₆) δ 8.82 (s, 1H), 7.61 (d,

J = 5.9 Hz, 1H), 7.36−7.32 (m, 2H), 6.27 (d, J = 1.9 Hz, 1H), 5.21 (s,

2H), 3.85 (s, 3H). ¹³C NMR (150 MHz, DMSO) δ 152.6, 148.0,

137.6, 137.2, 135.0, 122.9, 116.7, 107.7, 106.3, 41.1, 36.6. HRMS

(ESI): m/z calcd for C₁₁H₁₁N₆O₃[M + H]⁺, 275.0887; found,

275.0897.

7-((1H-Benzo[d]imidazol-2-yl)methyl)-2-nitroimidazo[1,2-a]-

pyrazin-8(7H)-one (26e). 2-Nitroimidazo[1,2-a]pyrazin-8(7H)-one

16 (60 mg, 0.333 mmol, 1 equiv) was reacted with 2-(chloromethyl)-

1H-benzo[d]imidazole 24 (83.2 mg, 0.500 mmol, 1.5 equiv) under

microwave conditions at 90 °C (20 min x 2) according to the general

procedure method B. After the reaction was completed as analyzed by

LC-MS, the mixture was diluted with distilled water (15 mL) and the

precipitate was collected by ﬁltration. The crude product was puriﬁed

by C18-reversed phase silica (Grace Reveleris X2, A: 0.1% TFA in

water, B: 0.1% TFA in ACN, 0−100% B) to give a major product, 7-

((1H-benzo[d]imidazol-2-yl)methyl)-2-nitroimidazo[1,2-a]pyrazin-

8(7H)-one 26e, as a cream color solid (43 mg, 42%) and a minor

product, 7-((1-((1H-benzo[d]imidazol-2-yl)methyl)-1H-benzo[d]-

imidazol-2-yl)methyl)-2-nitroimidazo[1,2-a]pyrazin-8(7H)-one as a

cream color solid (35 mg, 24%). Major product LC-MS: R_t= 2.16

1

min, 99 Abs % @ 254 nm, [M + H]⁺= 311.1; H NMR (600 MHz,

DMSO-d₆) 8.87 (s, 1H), 7.66 (d, J = 5.9 Hz, 1H), 7.51 (dt, J = 5.9,

3.5 Hz, 2H), 7.48 (d, J = 5.9 Hz, 1H), 7.20−7.14 (m, 2H), 5.39 (s,

2H). ¹³C NMR (150 MHz, DMSO-d₆) δ 152.9, 149.5, 148.0, 135.0,

124.2, 121.9, 116.7, 107.3, 45.2. HRMS (ESI): m/z calcd for

C₁₄H₁₁N₆O₃[M + H]⁺, 311.0887; found, 311.0900. Minor product

LC-MS: R_t= 2.50 min, 99 Abs % @ 254 nm, [M + H]⁺= 441.2.

7-((1-Methyl-1H-benzo[d]imidazol-2-yl)methyl)-2-nitroimidazo-

[1,2-a]pyrazin-8(7H)-one (26f). 2-Nitroimidazo[1,2-a]pyrazin-

8(7H)-one 16 (100 mg, 0.555 mmol, 1 equiv) was reacted with 2-

(chloromethyl)-1-methyl-1H-benzo[d]imidazole 25 (150 mg, 0.833

mmol, 1.5 equiv) under microwave conditions at 90 °C for 20 min

according to the general procedure method B. After the reaction was

completed as analyzed by LC-MS, the mixture was diluted with

4-Nitro-N-(pyrazin-2-ylmethyl)-1H-iimidazole-2-carboxamide

(27i). A mixture of acid chloride crude solid (150 mg, 0.855 mmol, 1

equiv) and triethylamine (238 μL, 1.71 mmol, 2 equiv) was reacted

with aminomethylpyrazine (112 mg, 1.03 mmol, 1.2 equiv) according

to general procedure method E. After completion of reaction, volatiles

O

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were removed in vacuo before purifying over C18-reversed phase

silica (Grace Reveleris X2, A: 0.1% TFA in water, B: 0.1% TFA in

ACN, 0−100% B) to give the ﬁnal product, 4-nitro-N-(pyrazin-2-

ylmethyl)-1H-imidazole-2-carboxamide 27i, as a beige solid (121 mg,

57%). LC-MS: R_t= 1.99 min, 99 Abs % @ 254 nm, [M + H]⁺=

equiv) according to the general procedure method F. After

completion of reaction, the mixture was diluted with distilled water

(28 mL) and extracted with EtOAc (3 × 28 mL). The organic layer

was collected, washed with brine (28 mL), and dried with MgSO₄.

Volatiles were removed in vacuo and the crude product was puriﬁed

over silica gel by MPLC (Biotage Isolera, 15−100% pet. spirit/

EtOAc) to give 1-(2,2-diethoxyethyl)-4-nitro-N-(pyrazin-2-ylmethyl)-

1H-imidazole-2-carboxamide 28i as a light gray solid (38 mg, 19%).

LC-MS: R_t= 2.72 min, 99 Abs % @ 254 nm, [M + Na]⁺= 365.1. ¹H

NMR (600 MHz, CDCl₃) δ 8.64 (d, J = 1.4 Hz, 1H), 8.57 (ddd, J =

6.9, 2.5, 1.5 Hz, 1H), 8.53 (d, J = 2.6 Hz, 1H), 8.16 (d, J = 5.9 Hz,

1H), 7.93 (s, 1H), 4.76 (d, J = 5.9 Hz, 2H), 4.73 (t, J = 4.9 Hz, 1H),

4.66 (d, J = 4.9 Hz, 2H), 3.75 (dq, J = 9.4, 7.1 Hz, 2H), 3.53 (dq, J =

9.4, 7.0 Hz, 2H), 1.18 (t, J = 7.0 Hz, 6H).

1-(2,2-Diethoxyethyl)-4-nitro-N-(quinolin-6-ylmethyl)-1H-imida-

zole-2-carboxamide (28j). 4-Nitro-N-(quinolin-6-ylmethyl)-1H-imi-

dazole-2-carboxamide (180 mg, 0.606 mmol, 1 equiv) was reacted

with bromoacetaldehyde diethyl acetal (137 μL, 0.908 mmol, 1.5

equiv) according to the general procedure method F. After

completion of reaction, the mixture was diluted with distilled water

(36 mL) and extracted with EtOAc (3 × 36 mL). The organic layer

was collected, washed with brine (22 mL), and dried with MgSO₄

before removing the volatiles in vacuo. The crude product looked

clean when analyzed using LC-MS, and therefore was used in the next

reaction without further purifying (204 mg, 81%). LC-MS: R_t= 2.72

min, 91 Abs % @ 254 nm, [M + Na]⁺= 414.2. ¹H NMR (600 MHz,

CDCl₃) δ 8.94 (dd, J = 4.2, 1.7 Hz, 1H), 8.20−8.08 (m, 2H), 7.99−

7.87 (m, 2H), 7.83−7.75 (m, 1H), 7.70 (dd, J = 8.7, 2.0 Hz, 1H), 7.44

(dd, J = 8.3, 4.2 Hz, 1H), 4.86−4.76 (m, 3H), 4.71 (d, J = 4.8 Hz,

2H), 3.77 (dq, J = 9.4, 7.0 Hz, 2H), 3.56 (dq, J = 9.4, 7.1 Hz, 2H),

1.20 (t, J = 7.0 Hz, 6H).

N-(Benzo[d][1,3]dioxol-5-ylmethyl)-1-(2,2-diethoxyethyl)-4-

nitro-1H-imidazole-2-carboxamide (28k). N-(Benzo[d][1,3]dioxol-

5-ylmethyl)-4-nitro-1H-imidazole-2-carboxamide (110 mg, 0.379

mmol, 1 equiv) was reacted with bromoacetaldehyde diethyl acetal

(85.5 μL, 0.569 mmol, 1.5 equiv) according to the general procedure

method F. After completion of reaction, the mixture was diluted with

distilled water (22 mL) and extracted with EtOAc (3 × 22 mL). The

organic layer was collected, washed with brine (22 mL), and dried

with MgSO₄before removing the volatiles in vacuo. The crude

product looked clean when analyzed using LC-MS, and therefore was

used in the next reaction without further purifying. LC-MS: R_t= 2.95

min, 99 Abs % @ 254 nm, [M + Na]⁺= 429.1. ¹H NMR (600 MHz,

DMSO-d₆) δ 9.37 (t, J = 6.4 Hz, 1H), 8.48 (s, 1H), 6.90 (d, J = 1.6

Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H), 6.79 (dd, J = 7.9, 1.6 Hz, 1H), 5.97

(s, 2H), 4.77 (t, J = 5.1 Hz, 1H), 4.60 (d, J = 5.1 Hz, 2H), 4.31 (d, J =

6.3 Hz, 2H), 3.60 (dq, J = 9.8, 7.0 Hz, 2H), 3.40 (dq, J = 9.8, 7.1 Hz,

2H), 1.01 (t, J = 7.0 Hz, 6H).

1

249.1; H NMR (600 MHz, DMSO-d₆) δ 9.47 (t, J = 6.1 Hz, 1H),

8.68−8.56 (m, 2H), 8.54 (d, J = 2.6 Hz, 1H), 8.48 (s, 1H), 4.62 (d, J

= 6.1 Hz, 2H). ¹³C NMR (150 MHz, DMSO) δ 157.5, 153.5, 146.7,

143.9, 143.4, 143.2, 139.3, 121.6, 42.3.

4-Nitro-N-(quinolin-6-ylmethyl)-1H-imidazole-2-carboxamide

(27j). A mixture of acid chloride crude solid (310 mg, 1.77 mmol, 1

equiv) and triethylamine (492 μL, 3.53 mmol, 2 equiv) was reacted

with 6-aminomethylquinoline (335 mg, 290 μL, 2.12 mmol, 1.2

equiv) according to general procedure method E. After completion of

reaction, volatiles were removed in vacuo before purifying over C18-

reversed phase silica (Grace Reveleris X2, A: 0.1% TFA in water, B:

0.1% TFA in ACN, 0−100% B) to give the ﬁnal product 4-nitro-N-

(quinolin-6-ylmethyl)-1H-imidazole-2-carboxamide 27j, as a white

solid (612 mg, 91%, contained 0.7 equivalent of TFA). LC-MS: R_t=

1

2.15 min, 99 Abs % @ 254 nm, [M + H]⁺= 298.1; H NMR (600

MHz, DMSO-d₆) δ 9.63 (t, J = 6.3 Hz, 1H), 9.00 (dd, J = 4.5, 1.7 Hz,

1H), 8.62 (d, J = 8.3 Hz, 1H), 8.48 (d, J = 1.3 Hz, 1H), 8.07 (d, J =

8.7 Hz, 1H), 7.98 (d, J = 1.9 Hz, 1H), 7.87 (dd, J = 8.7, 2.0 Hz, 1H),

7.69 (dd, J = 8.3, 4.5 Hz, 1H), 4.67 (d, J = 6.2 Hz, 2H). ¹³C NMR

(150 MHz, DMSO) δ 158.2, 158.0, 157.4, 148.7, 146.8, 139.5, 138.4,

130.9, 127.9, 126.6, 125.8, 121.8, 121.6, 42.20.

N-(Benzo[d][1,3]dioxol-5-ylmethyl)-4-nitro-1H-imidazole-2-car-

boxamide (27k). A mixture of acid chloride crude solid (150 mg,

0.855 mmol, 1 equiv) and triethylamine (238 μL, 1.71 mmol, 2 equiv)

was reacted with 1,3-benzodioxole-5-methanamine (155 mg, 128 μL,

1.03 mmol, 1.2 equiv) according to general procedure method E.

After completion of reaction, volatiles were removed in vacuo before

purifying over C18-reversed phase silica (Grace Reveleris X2, A: 0.1%

TFA in water, B: 0.1% TFA in ACN, 0−100% B) to give the ﬁnal

product, N-(benzo[d][1,3]dioxol-5-ylmethyl)-4-nitro-1H-imidazole-

2-carboxamide 27k, as a white solid (136 mg, 55%). LC-MS: R_t=

1

2.61 min, 98 Abs % @ 254 nm, [M − H]⁻= 288.9. H NMR (600

MHz, DMSO-d₆) δ 9.37 (t, J = 6.4 Hz, 1H), 8.45 (s, 1H), 6.90 (d, J =

1.6 Hz, 1H), 6.84 (d, J = 7.9 Hz, 1H), 6.79 (dd, J = 7.9, 1.6 Hz, 1H),

5.97 (s, 2H), 4.33 (d, J = 6.3 Hz, 2H). ¹³C NMR (150 MHz, DMSO-

d₆) δ 157.0, 147.1, 146.7, 146.1, 139.6, 132.9, 121.5, 120.8, 108.2,

108.0, 100.8, 42.1.

1-(2,2-Diethoxyethyl)-4-nitro-N-(thiazol-2-ylmethyl)-1H-imida-

zole-2-carboxamide (28b). 4-Nitro-N-(thiazol-2-ylmethyl)-1H-imi-

dazole-2-carboxamide (140 mg, 0.553 mmol, 1 equiv) was reacted

with bromoacetaldehyde diethyl acetal (125 μL, 0.829 mmol, 1.5

equiv) according to the general procedure method F. After

completion of reaction, the mixture was diluted with distilled water

(28 mL) and extracted with EtOAc (3 × 28 mL). The organic layer

was collected, washed with brine (28 mL), and dried with MgSO₄.

Volatiles were removed in vacuo and the crude product was puriﬁed

over silica gel by MPLC (Biotage Isolera, 15−100% pet. spirit/

EtOAc) to give 1-(2,2-diethoxyethyl)-4-nitro-N-(thiazol-2-ylmethyl)-

1H-imidazole-2-carboxamide 28b as a beige solid (94 mg, 46%). LC-

MS: R_t= 2.79 min, 93 Abs % @ 254 nm, [M + Na]⁺= 370.1.

1-(2,2-Diethoxyethyl)-5-nitro-N-(pyridin-3-ylmethyl)-1H-imida-

zole-2-carboxamide (28g). 5-Nitro-N-(pyridin-3-ylmethyl)-1H-imi-

dazole-2-carboxamide (120 mg, 0.485 mmol, 1 equiv) was reacted

with bromoacetaldehyde diethyl acetal (110 μL, 0.728 mmol, 1.5

equiv) according to the general procedure method F. After

completion of reaction, the mixture was diluted with distilled water

(24 mL) and extracted with EtOAc (3 × 24 mL). The organic layer

was collected, washed with brine (24 mL), and dried with MgSO₄

before removing the volatiles in vacuo. The crude product was used in

the next reaction without further purifying (175 mg, 99%). LC-MS: R_t

= 2.43 min, 79 Abs % @ 254 nm, [M + H]⁺= 364.2.

2-Nitro-7-(thiazol-2-ylmethyl)imidazo[1,2-a]pyrazin-8(7H)-one

(26b). 1-(2,2-Diethoxyethyl)-4-nitro-N-(thiazol-2-ylmethyl)-1H-imi-

dazole-2-carboxamide 28b (59 mg, 0.160 mmol, 1 equiv) was reacted

according to the general procedure method G. After completion of

reaction, the volatiles were evaporated in vacuo followed by

recrystallization using DCM/MeOH (hot slurry) to yield the desired

product, 2-nitro-7-(thiazol-2-ylmethyl)imidazo[1,2-a]pyrazin-8(7H)-

one 26b, as a yellow solid (26 mg, 58%): LC-MS: R_t= 2.46 min, 99

1

Abs % @ 254 nm, [M + H]⁺= 278.1; H NMR (600 MHz, DMSO-

d₆) δ 8.84 (s, 1H), 7.77 (d, J = 3.2 Hz, 1H), 7.74 (d, J = 3.3 Hz, 1H),

7.64 (d, J = 5.9 Hz, 1H), 7.50 (d, J = 5.9 Hz, 1H), 5.46 (s, 2H). ¹³C

NMR (150 MHz, DMSO) δ 164.1, 152.5, 148.1, 142.4, 134.7, 123.7,

121.4, 116.9, 107.5, 48.0. HRMS (ESI): m/z calcd for C₁₀H₈N₅O₃S

[M + H]⁺, 278.0342; found, 278.0356.

2-Nitro-7-(pyridin-3-ylmethyl)imidazo[1,2-a]pyrazin-8(7H)-one

(26g). 1-(2,2-Diethoxyethyl)-5-nitro-N-(pyridin-3-ylmethyl)-1H-imi-

dazole-2-carboxamide 28g (175 mg, 0.482 mmol, 1 equiv) was

reacted according to the general procedure method G. Volatiles were

removed in vacuo, and the crude product was puriﬁed by C18-

reversed phase silica (Grace Reveleris X2, A: 0.1% TFA in water, B:

0.1% TFA in ACN, 0−100% B), then re-puriﬁed by HPLC (Gilson,

A: 0.1% TFA in water, B: 0.1% TFA in ACN, 0−50% B) to give ﬁnal

1-(2,2-Diethoxyethyl)-4-nitro-N-(pyrazin-2-ylmethyl)-1H-imida-

zole-2-carboxamide (28i). 4-Nitro-N-(pyrazin-2-ylmethyl)-1H-imi-

dazole-2-carboxamide (137 mg, 0.552 mmol, 1 equiv) was reacted

with bromoacetaldehyde diethyl acetal (125 μL, 0.828 mmol, 1.5

P

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product, 2-nitro-7-(pyridin-3-ylmethyl)imidazo[1,2-a]pyrazin-8(7H)-

one 26g, as a light brown solid (17 mg, 13%). LC-MS: R_t= 1.61 min,

Hz, 2H). ¹³C NMR (150 MHz DMSO-d₆) δ 192.7, 148.6, 144.3,

138.1, 135.3, 130.2, 129.2, 127.6, 121.6, 120.1 (J = 256.6 Hz).

4′-Methyl-[1,1′-biphenyl]-4-carbaldehyde (31c). A mixture of 4-

bromobenzaldehyde (550 mg, 2.97 mmol), p-tolylboronic acid (1.1

equiv, 445 mg, 3.27 mmol), K₂CO₃(1.23 g, 8.92 mmol), and

tetrakis(triphenylphosphine)palladium (0) (275 mg, 0.238 mmol) in

THF (8 mL) and distilled water (3 mL) was reacted according to the

general procedure method H. After completion of reaction, the

mixture was diluted with distilled water and extracted with EtOAc (x

3). The combined organic layers were washed with brine, dried over

MgSO₄, and evaporated in vacuo. The crude product was puriﬁed

over silica gel by MPLC (Biotage Isolera, 2−20% pet. spirit/EtOAc)

to give ﬁnal product, 4′-methyl-[1,1′-biphenyl]-4-carbaldehyde 31c as

a white solid (355 mg, 61% yield): LC-MS: R_t= 3.07 min, 99 Abs %

1

97 Abs % @ 254 nm, [M + H]⁺= 272.1; H NMR (600 MHz,

DMSO-d₆) δ 8.82 (s, 1H), 8.62 (d, J = 2.2 Hz, 1H), 8.51 (dd, J = 4.8,

1.6 Hz, 1H), 7.77 (dt, J = 7.9, 2.0 Hz, 1H), 7.62 (d, J = 5.9 Hz, 1H),

7.49 (d, J = 5.9 Hz, 1H), 7.38 (dd, J = 7.9, 4.8 Hz, 1H), 5.17 (s, 2H).

¹³C NMR (150 MHz, DMSO-d₆) δ 152.9, 149.1, 149.0, 148.0, 135.6,

135.2, 132.2, 123.7, 123.5, 116.7, 107.6, 48.2. HRMS (ESI): m/z

calcd for C₁₂H₁₀N₅O₃[M + H]⁺, 272.0789; found, 272.0778.

2-Nitro-7-(pyrazin-2-ylmethyl)imidazo[1,2-a]pyrazin-8(7H)-one

(26i). 1-(2,2-Diethoxyethyl)-4-nitro-N-(pyrazin-2-ylmethyl)-1H-imi-

dazole-2-carboxamide 28i (37 mg, 0.102 mmol, 1 equiv) was reacted

according to the general procedure method G. After completion of

reaction, the volatiles were evaporated in vacuo followed by

recrystallization using DCM/MeOH (hot slurry) to yield the desired

product, 2-nitro-7-(pyrazin-2-ylmethyl)imidazo[1,2-a]pyrazin-8(7H)-

one 26i, as a gray solid (10 mg, 35%): LC-MS: R_t= 2.19 min, 99 Abs

% @ 254 nm, [M + H]⁺= 273.1; ¹H NMR (600 MHz, DMSO-d₆) δ

8.84 (s, 1H), 8.74 (d, J = 1.4 Hz, 1H), 8.61−8.56 (m, 2H), 7.63 (d, J

= 5.9 Hz, 1H), 7.48 (d, J = 5.8 Hz, 1H), 5.32 (s, 2H). ¹³C NMR (150

MHz, DMSO) δ 152.8, 151.2, 148.0, 144.1, 143.8, 135.0, 124.4,

116.7, 109.5, 107.1, 50.1. HRMS (ESI): m/z calcd for C₁₁H₉N₆O₃[M

+ H]⁺, 273.0731; found, 273.0746.

1

@ 254 nm, [M + H]⁺= 197.2; H NMR (600 MHz, DMSO-d₆) δ

10.04 (s, 1H), 7.98 (d, J = 8.0 Hz, 2H), 7.89 (d, J = 8.0 Hz, 2H), 7.67

(d, J = 7.9 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 2.36 (s, 3H). ¹³C NMR

(150 MHz, DMSO-d₆) δ 192.7, 145.8, 138.2, 135.8, 134.8, 130.1,

129.7, 127.0, 126.9, 20.7.

3′-(Triﬂuoromethoxy)-[1,1′-biphenyl]-4-carbaldehyde (31d). A

mixture of 4-bromobenzaldehyde (550 mg, 2.97 mmol), (3-

(triﬂuoromethoxy)phenyl)boronic acid (1.1 equiv, 673 mg, 3.27

mmol), K₂CO₃(1.23 g, 8.92 mmol), and tetrakis-

(triphenylphosphine)palladium (0) (275 mg, 0.238 mmol) in THF

(8 mL) and distilled water (3 mL) was reacted according to the

general procedure method H. After completion of reaction, the

mixture was ﬁltered through celite and the ﬁltrate was evaporated in

vacuo. The crude product was puriﬁed over silica gel by MPLC

(Biotage Isolera, 2−20% pet. spirit/EtOAc) to give ﬁnal product, 3′-

(triﬂuoromethoxy)-[1,1′-biphenyl]-4-carbaldehyde 31d, in a colorless

gel form (710 mg, 90% yield): LC-MS: R_t= 3.18 min, 99 Abs % @

254 nm, [M + H]⁺= 267.1; ¹H NMR (400 MHz, DMSO-d₆) δ 10.08

(s, 1H), 8.06−7.93 (m, 4H), 7.83 (ddt, J = 7.8, 1.9, 0.9 Hz, 1H), 7.76

(s, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.50−7.42 (m, 1H).

6-Phenylnicotinaldehyde (31f). A mixture of 6-bromonicotinalde-

hyde (550 mg, 2.96 mmol), phenylboronic acid (1.1 equiv, 397 mg,

3.25 mmol), K₂CO₃(1.23 g, 8.87 mmol), tetrakis-

(triphenylphosphine)palladium (0) (273 mg, 0.237 mmol), THF (8

mL), and distilled water (3 mL) was reacted according to the general

procedure method H. After completion of reaction, the mixture was

diluted with distilled water and extracted with EtOAc (x 3). The

combined organic layers were washed with brine, dried over MgSO₄,

and evaporated in vacuo. The crude product was puriﬁed over silica

gel by MPLC (Biotage Isolera, 2−20% pet. spirit/EtOAc) to give the

ﬁnal product, 6-phenylnicotinaldehyde 31f, as a white solid (427 mg,

79% yield): LC-MS: R_t= 2.65 min, 99 Abs % @ 254 nm, [M + H]⁺=

184.1; ¹H NMR (600 MHz, DMSO-d₆) δ 10.14 (s, 1H), 9.16 (p, J =

1.3 Hz, 1H), 8.35−8.26 (m, 1H), 8.23−8.18 (m, 3H), 7.54 (dtt, J =

8.6, 5.4, 1.7 Hz, 3H). ¹³C NMR (150 MHz DMSO-d₆) δ 192.0, 160.4,

151.7, 137.4, 137.2, 130.4, 129.9, 129.0, 127.3, 120.5.

2-Nitro-7-(quinolin-6-ylmethyl)imidazo[1,2-a]pyrazin-8(7H)-one

(26j). 1-(2,2-Diethoxyethyl)-4-nitro-N-(quinolin-6-ylmethyl)-1H-imi-

dazole-2-carboxamide 28j (200 mg, 0.484 mmol, 1 equiv) was reacted

according to the general procedure method G. After completion of

reaction, the volatiles were evaporated in vacuo. The crude product

was then puriﬁed over silica gel by MPLC (Biotage Isolera, 1−25%

DCM/MeOH), followed by recrystallization in DCM/MeOH (hot

slurry) to give 2-nitro-7-(quinolin-6-ylmethyl)imidazo[1,2-a]pyrazin-

8(7H)-one 26j as a light brown solid (129 mg, 83%): LC-MS: R_t=

1

2.21 min, 99 Abs % @ 254 nm, [M + H]⁺= 322.1; H NMR (600

MHz, DMSO-d₆) δ 8.89 (dd, J = 4.1, 1.7 Hz, 1H), 8.84 (s, 1H), 8.33

(ddd, J = 8.4, 1.8, 0.8 Hz, 1H), 8.02 (d, J = 8.7 Hz, 1H), 7.90 (d, J =

2.0 Hz, 1H), 7.75 (dd, J = 8.7, 2.1 Hz, 1H), 7.64 (d, J = 5.9 Hz, 1H),

7.53 (dd, J = 8.3, 4.1 Hz, 1H), 7.49 (d, J = 5.8 Hz, 1H), 5.34 (s, 2H).

¹³C NMR (150 MHz, DMSO) δ 152.9, 150.7, 148.0, 147.2, 135.9,

135.2, 134.7, 129.4, 129.2, 127.7, 126.3, 123.6, 121.8, 116.7, 107.5,

50.1. HRMS (ESI): m/z calcd for C₁₆H₁₂N₅O₃[M + H]⁺, 322.0935;

found, 322.0932.

7-(Benzo[d][1,3]dioxol-5-ylmethyl)-2-nitroimidazo[1,2-a]-

pyrazin-8(7H)-one (26k). N-(Benzo[d][1,3]dioxol-5-ylmethyl)-1-

(2,2-diethoxyethyl)-4-nitro-1H-imidazole-2-carboxamide 28k (150

mg, 0.369 mmol, 1 equiv) was reacted according to the general

procedure method G. After completion of reaction, the volatiles were

evaporated in vacuo followed by recrystallization using DCM/MeOH

(hot slurry) to yield the desired product, 7-(benzo[d][1,3]dioxol-5-

ylmethyl)-2-nitroimidazo[1,2-a]pyrazin-8(7H)-one 26k, as a yellow

solid (71 mg, 61%): LC-MS: R_t= 2.66 min, 99 Abs % @ 254 nm, [M

+ H]⁺= 315.1; ¹H NMR (600 MHz, DMSO-d₆) δ 8.80 (s, 1H), 7.58

(d, J = 5.9 Hz, 1H), 7.39 (d, J = 5.9 Hz, 1H), 6.96 (d, J = 1.4 Hz, 1H),

6.88 (d, J = 1.6 Hz, 2H), 5.99 (s, 2H), 5.02 (s, 2H). ¹³C NMR (150

MHz, DMSO) δ 152.7, 148.0, 147.4, 146.9, 135.1, 130.2, 123.3,

121.6, 116.6, 108.5, 108.3, 107.4, 101.1, 49.9. HRMS (ESI): m/z

calcd for C₁₄H₁₁N₄O₅[M + H]⁺, 315.0724; found, 315.0715.

4′-(Triﬂuoromethoxy)-[1,1′-biphenyl]-4-carbaldehyde (31b). A

mixture of 4-bromobenzaldehyde (1.10 g, 5.95 mmol), (4-

(triﬂuoromethoxy)phenyl)boronic acid (1.1 equiv, 1.35 g, 6.54

mmol), K₂CO₃(2.47 g, 17.8 mmol), and tetrakis-

(triphenylphosphine)palladium (0) (0.547 g, 0.476 mmol) in THF

(16 mL) and distilled water (6 mL) was reacted according to the

general procedure method H. After completion of reaction, the

mixture was ﬁltered through celite and the ﬁltrate was evaporated in

vacuo. The crude product was puriﬁed over silica gel by MPLC

(Biotage Isolera, 2−20% pet. spirit/EtOAc) to give ﬁnal product, 4′-

(triﬂuoromethoxy)-[1,1′-biphenyl]-4-carbaldehyde 31b, as a white

solid (1.31 g, 83% yield): LC-MS: R_t= 3.18 min, 99 Abs % @ 254

6-(4-(Triﬂuoromethoxy)phenyl)nicotinaldehyde (31g). A mixture

of 6-bromonicotinaldehyde (550 mg, 2.96 mmol), (4-

(triﬂuoromethoxy)phenyl)boronic acid (1.1 equiv, 670 mg, 3.25

mmol), K₂CO₃(1.23 g, 8.87 mmol), and tetrakis-

(triphenylphosphine)palladium (0) (273 mg, 0.237 mmol) in THF

(8 mL) and distilled water (3 mL) was reacted according to the

general procedure method H. After completion of reaction, the

mixture was diluted with distilled water and extracted with EtOAc (x

3). The combined organic layers were washed with brine, dried over

MgSO₄, and evaporated in vacuo. The crude product was puriﬁed

over silica gel by MPLC (Biotage Isolera, 2−20% pet. spirit/EtOAc)

to give the ﬁnal product, 6-(4-(triﬂuoromethoxy)phenyl)-

nicotinaldehyde 31g, as a white solid (560 mg, 71% yield): LC-MS:

1

R_t= 2.99 min, 98 Abs % @ 254 nm, [M + H]⁺= 268.1; H NMR

(600 MHz, DMSO-d₆) δ ¹H NMR (600 MHz, DMSO-d₆) δ 10.15 (s,

1H), 9.17 (dd, J = 2.2, 0.9 Hz, 1H), 8.37−8.29 (m, 3H), 8.25 (d, J =

8.2 Hz, 1H), 7.56−7.51 (m, 2H). ¹³C NMR (150 MHz, DMSO-d₆) δ

192.0, 158.9, 151.6, 149.8, 137.4, 136.6, 130.1, 129.4, 121.3, 120.8,

120.1 (J = 256.4 Hz).

1

nm, [M + H]⁺= 267.1; H NMR (600 MHz, DMSO-d₆) δ 10.07 (s,

1H), 8.04−7.99 (m, 2H), 7.97−7.88 (m, 4H), 7.51 (dq, J = 7.7, 1.0

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c01372

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

6-(p-Tolyl)nicotinaldehyde (31h). A mixture of 6-bromonicotinal-

dehyde (550 mg, 2.96 mmol), p-tolylboronic acid (1.1 equiv, 442 mg,

3.25 mmol), K₂CO₃(1.23 g, 8.87 mmol), and tetrakis-

(triphenylphosphine)palladium (0) (273 mg, 0.237 mmol) in THF

(8 mL) and distilled water (3 mL) was reacted according to the

general procedure method H. After completion of reaction, the

mixture was diluted with distilled water and extracted with EtOAc (x

3). The combined organic layers were washed with brine, dried over

MgSO₄, and evaporated in vacuo. The crude product was puriﬁed

over silica gel by MPLC (Biotage Isolera, 2−20% pet. spirit/EtOAc)

to give the ﬁnal product, 6-(p-tolyl)nicotinaldehyde 31h, as a white

solid (391 mg, 67% yield): LC-MS: R_t= 2.80 min, 99 Abs % @ 254

1H), 9.21 (dd, J = 2.4, 0.8 Hz, 1H), 9.11−9.07 (m, 1H), 8.56−8.52

(m, 1H), 8.00−7.94 (m, 2H), 7.54 (d, J = 8.3 Hz, 2H). ¹³C NMR

(150 MHz DMSO-d₆) δ 192.4, 152.5, 150.1, 148.7, 135.2, 134.6,

134.3, 131.2, 129.2, 121.7, 120.1 (J = 256.0 Hz).

5-(p-Tolyl)nicotinaldehyde (31l). A mixture of 5-bromonicotinal-

dehyde (400 mg, 2.15 mmol), p-tolylboronic acid (1.1 equiv, 322 mg,

2.37 mmol), K₂CO₃(892 mg, 6.45 smmol), tetrakis-

(triphenylphosphine)palladium (0) (199 mg, 0.172 mmol), THF

(5.8 mL), and distilled water (2.2 mL) was reacted according to the

general procedure method H. After completion of reaction, the

mixture was diluted with distilled water and extracted with EtOAc (x

3). The combined organic layers were washed with brine, dried over

MgSO₄, and evaporated in vacuo. The crude product was puriﬁed

over silica gel by MPLC (Biotage Isolera, 12−100% pet. spirit/

EtOAc) to give the ﬁnal product, 5-(p-tolyl)nicotinaldehyde 31l, as a

yellow solid (240 mg, 57% yield): LC-MS: R_t= 2.71 min, 95 Abs % @

254 nm, [M + H]⁺= 198.2; ¹H NMR (600 MHz, DMSO-d₆) δ 10.19

(s, 1H), 9.16 (d, J = 2.4 Hz, 1H), 9.03 (d, J = 1.9 Hz, 1H), 8.48 (dd, J

= 2.4, 1.9 Hz, 1H), 7.77−7.70 (m, 2H), 7.39−7.33 (m, 2H), 2.37 (s,

3H). ¹³C NMR (150 MHz DMSO-d₆) δ 192.6, 152.2, 149.4, 138.3,

135.8, 133.7, 132.9, 131.3, 129.9, 126.9, 20.7.

1

nm, [M + H]⁺= 198.1; H NMR (600 MHz, DMSO-d₆) δ 10.12 (s,

1H), 9.13 (dd, J = 2.2, 0.9 Hz, 1H), 8.29 (dd, J = 8.3, 2.2 Hz, 1H),

8.19−8.15 (m, 1H), 8.13−8.08 (m, 2H), 7.38−7.33 (m, 2H), 2.38 (s,

3H). ¹³C NMR (150 MHz, DMSO-d₆) δ 191.9, 160.4, 151.7, 140.3,

137.0, 134.7, 129.7, 129.6, 127.2, 120.1, 20.9.

6-(3-(Triﬂuoromethoxy)phenyl)nicotinaldehyde (31i). A mixture

of 6-bromonicotinaldehyde (550 mg, 2.96 mmol), (3-

(triﬂuoromethoxy)phenyl)boronic acid (1.1 equiv, 670 mg, 3.25

mmol), K₂CO₃(1.23 g, 8.87 mmol), and tetrakis-

(triphenylphosphine)palladium (0) (273 mg, 0.237 mmol) in THF

(8 mL) and distilled water (3 mL) was reacted according to the

general procedure method H. After completion of reaction, the

mixture was diluted with distilled water and extracted with EtOAc (x

3). The combined organic layers were washed with brine, dried over

MgSO₄, and evaporated in vacuo. The crude product was puriﬁed

over silica gel by MPLC (Biotage Isolera, 2−20% pet. spirit/EtOAc)

to give the ﬁnal product, 6-(3-(triﬂuoromethoxy)phenyl)-

nicotinaldehyde 31i, as a white solid (377 mg, 48% yield): LC-MS:

5-(3-(Triﬂuoromethoxy)phenyl)nicotinaldehyde (31m). A mix-

ture of 5-bromonicotinaldehyde (400 mg, 2.15 mmol), (3-

(triﬂuoromethoxy)phenyl)boronic acid (1.1 equiv, 487 mg, 2.37

mmol), K₂CO₃(892 mg, 6.45 mmol), tetrakis(triphenylphosphine)-

palladium (0) (199 mg, 0.172 mmol), THF (5.8 mL), and distilled

water (2.2 mL) was reacted according to the general procedure

method H. After completion of reaction, the mixture was diluted with

distilled water and extracted with EtOAc (x 3). The combined organic

layers were washed with brine, dried over MgSO₄, and evaporated in

vacuo. The crude product was puriﬁed over silica gel by MPLC

(Biotage Isolera, 7−60% pet. spirit/EtOAc) to give the ﬁnal product,

5-(3-(triﬂuoromethoxy)phenyl)nicotinaldehyde 31m, as a cream

color solid (331 mg, 58% yield): LC-MS: R_t= 2.94 min, 98 Abs %

1

R_t= 3.01 min, 99 Abs % @ 254 nm, [M + H]⁺= 268.0; H NMR

(600 MHz, DMSO-d₆) δ 10.16 (s, 1H), 9.19 (d, J = 2.1 Hz, 1H), 8.36

(dd, J = 8.2, 2.1 Hz, 1H), 8.31 (d, J = 8.2 Hz, 1H), 8.25 (dt, J = 8.0,

1.1 Hz, 1H), 8.18 (d, J = 1.9 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.57−

7.51 (m, 1H). ¹³C NMR (150 MHz, DMSO-d₆) δ 192.0, 158.4, 151.6,

149.0, 139.7, 137.5, 131.1, 130.5, 126.3, 122.7, 121.0, 120.2 (J = 256.6

Hz), 119.5.

1

@ 254 nm, [M + H]⁺= 268.1; H NMR (600 MHz, DMSO-d₆) δ

10.20 (s, 1H), 9.23 (dd, J = 2.3, 0.9 Hz, 1H), 9.10 (d, J = 1.2 Hz, 1H),

8.58 (td, J = 2.2, 0.9 Hz, 1H), 7.92−7.86 (m, 2H), 7.72−7.65 (m,

1H), 7.49 (ddt, J = 8.2, 2.1, 1.1 Hz, 1H). ¹³C NMR (150 MHz

DMSO-d₆) δ 192.4, 152.6, 150.2, 149.1, 138.3, 134.7, 134.3, 131.2,

126.3, 121.1, 120.9, 120.1 (J = 256.4 Hz), 119.9.

5-(4-(Triﬂuoromethoxy)phenyl)picolinaldehyde (31j). A mixture

of 5-bromopicolinaldehyde (400 mg, 2.15 mmol), (4-