Design, synthesis and biological evaluation of diamino substituted cyclobut-3-ene-1,2-dione derivatives for the treatment of drug-resistant tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2020.112538

Mycobacterium tuberculosis (Mtb) ATP synthase is an important target for treating drug-resistant infections and sterilizing the bacteria, spurring intensive efforts to develop new TB therapeutics based on this target. In this work, four novel series including furan-2(5H)-ketone (3, 4), maleimide (5) and squaramide (6) derivatives were designed, respectively, through the strategy of scaffold morphing and hydrogen-bond introduction, using the selective Mtb ATP synthase inhibitor compound 2 as the lead compound. The result demonstrated that diamino substituted cyclobut-3-ene-1,2-dione compounds 6ab and 6ah displayed good to excellent in vitro anti-TB activities (MIC 0.452–0.963 μg/mL) with low cytotoxicity (IC₅₀ > 64 μg/mL). In addition, not only did compound 6ab show effective activity against clinically isolated resistant strains, it also revealed good druggability profiles including improved metabolic stability, no hERG channel inhibition potential, and acceptable oral bioavailability. The preliminary result of docking study and in vitro anti-bedaquiline-resistant strain test compared to compound 2 suggested that Mtb ATP synthase is most likely the target of compound 6ab. The structure-activity relationship laid a good foundation for the identification of novel squaramides as a potential treatment of drug-resistant tuberculosis.

Full text of DOI:10.1016/j.ejmech.2020.112538

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Design, synthesis and biological evaluation of diamino substituted cyclobut-3-ene-1,2-
dione derivatives for the treatment of drug-resistant tuberculosis
Peng Li, Bin Wang, Gang Li, Lei Fu, Dongfeng Zhang, Ziyun Lin, Haihong Huang, Yu
Lu
PII:
S0223-5234(20)30510-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112538
EJMECH 112538
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To appear in: European Journal of Medicinal Chemistry
Received Date: 30 March 2020
Revised Date: 24 May 2020
Accepted Date: 3 June 2020
Please cite this article as: P. Li, B. Wang, G. Li, L. Fu, D. Zhang, Z. Lin, H. Huang, Y. Lu, Design,
synthesis and biological evaluation of diamino substituted cyclobut-3-ene-1,2-dione derivatives
for the treatment of drug-resistant tuberculosis, European Journal of Medicinal Chemistry, https://
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Design, synthesis and biological evaluation of diamino
substituted cyclobut-3-ene-1,2-dione derivatives for the
treatment of drug-resistant tuberculosis
Peng Li1,2, Bin Wang3, Gang Li1,2, Lei Fu3, Dongfeng Zhang1,2, Ziyun Lin1,2, Haihong
Huang1,2,*, Yu Lu3,*
Table of Contents graphic

Design, synthesis and biological evaluation of diamino
substituted cyclobut-3-ene-1,2-dione derivatives for the
treatment of drug-resistant tuberculosis
Peng Li1,2, Bin Wang3, Gang Li1,2, Lei Fu3, Dongfeng Zhang1,2, Ziyun Lin1,2, Haihong
Huang1,2,*, Yu Lu3,*
1
Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation,
Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical
Sciences, 1 Xian Nong Tan Street, Beijing 100050, P. R. China
2 Chinese Academy of Medical Sciences Key Laboratory of Anti-DR TB Innovative Drug
Research, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of
Medical Sciences, 1 Xian Nong Tan Street, Beijing 100050, P. R. China
3
Beijing Key Laboratory of Drug Resistance Tuberculosis Research, Department of
Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing Chest
Hospital, Capital Medical University, 97 Ma Chang Street, Beijing 101149, P. R. China
*
Corresponding author. E-mail addresses: joyce@imm.ac.cn (H. Huang),
luyu4876@hotmail.com (Y. Lu).
ABSTRACT:
Mycobacterium tuberculosis (Mtb) ATP synthase is an important target for
treating drug-resistant infections and sterilizing the bacteria, spurring intensive efforts
to develop new TB therapeutics based on this target. In this work, four novel series
including furan-2(5H)-ketone (3, 4), maleimide (5) and squaramide (6) derivatives
were designed, respectively, through the strategy of scaffold morphing and
hydrogen-bond introduction, using the selective Mtb ATP synthase inhibitor
compound 2 as the lead compound. The result demonstrated that diamino substituted
cyclobut-3-ene-1,2-dione compounds 6ab and 6ah displayed good to excellent in
vitro anti-TB activities (MIC 0.452 ~ 0.963 μg/mL) with low cytotoxicity (IC50 > 64
μg/mL). In addition, not only did compound 6ab show effective activity against
clinically isolated resistant strains, it also revealed good druggability profiles

including improved metabolic stability, no hERG channel inhibition potential, and
acceptable oral bioavailability. The preliminary result of docking study and in vitro
anti-bedaquiline-resistant strain test compared to compound 2 suggested that Mtb
ATP synthase is most likely the target of compound 6ab. The structure-activity
relationship laid a good foundation for the identification of novel squaramides as a
potential treatment of drug-resistant tuberculosis.
KEYWORDS
Squaramide; Drug-resistant tuberculosis; Structure-activity relationship; ATP
synthase inhibitor.
1. Introduction
Tuberculosis (TB) is an infectious disease caused by Mycobacterium
tuberculosis (Mtb). Worldwide, TB is one of the top 10 causes of death and the
leading cause of death from a single infectious agent (above HIV/AIDS) [1]. In recent
years, the emergence and spread of multidrug-resistant (MDR) and extensively
drug-resistant (XDR) TB have presented great challenges to disease treatment and
prevention [2, 3]. Due to the high mortality and socioeconomic burden, drug-resistant
TB has become a serious social health and safety risk factor [4, 5]. In light of this
gloomy scenario, new anti-TB drugs to tackle drug-resistant infections are urgent
needed [6].
The energy metabolism pathway, essential for bacterial growth and survival, has
emerged as a novel target pathway in TB drug discovery recently [7]. As part of the
energy metabolism pathway, mycobacterial ATP synthase plays an important role for
the survival of bacteria under both replicating and non-replicating states [8]. Mtb ATP
synthase is a membrane-bound enzyme composed of multiple structural fragments
which utilize the electrochemical gradient across the membrane to produce ATP [9,
10]. This target has been validated for the development of promising new anti-TB
drugs [11], especially in complete sterilization [8]. One example of targeting bacterial
energetics for the treatment of drug-resistant TB [12] is bedaquiline (Fig. 1), the first

selective inhibitor of the Mtb ATP synthase. Bedaquiline was approved as the first
new anti-TB drug after rifampicin’s launch in the 1960s [13]. The unique mechanism
of action renders bedaquiline effective against drug-resistant TB [14, 15]. In addition,
arylquinoline compounds TBAJ-587 and TBAJ-876, as bedaquiline analogs, were in
preclinical studies with improved activity and reduced lipophilicity, exemplified by a
lower ClogP compared to bedaquiline [16, 17]. The purpose of increasing polarity
was to reduce cardiotoxicity related to bedaquiline’s high lipophilicity [18]. Despite
all the progress, new chemical entities are still needed to address the safety and
cross-resistance issues. High-throughput screening has provided an efficient way to
discover Mtb ATP synthase inhibitors with novel scaffolds distinct from
arylquinolines [19]. Recently, compound 1 and 2 (Fig. 1), obtained from a
high-throughput screening as non-quinolines Mtb ATP synthase inhibitors, were
under
lead
optimization
[20,
21].
Specifically,
compound
2
(monoamino-cyclobut-3-ene-1,2-dione) with very low lipophilicity displayed
efficiency in a mouse TB infection model and showed cross-resistance to some extent
against a bedaquiline-resistant mutant, which indicated that this compound might
occupy a different binding site of the ATP synthase compared to that of bedaquiline.
Therefore, it is worth conducting a lead optimization to identify novel Mtb ATP
synthase inhibitors based on compound 2 with lower lipophilicity and cardiotoxicity.
MeO
N
OMe
F
MeO
MeO
OH
OH
OH
Br
N
Br
N
Br
N
N
O
N
O
N
O
OMe
OMe
N
N
MeO
MeO
Bedaquiline
(Launched in 2012)
ClogP 7.25
TBAJ-587
(Preclinical)
ClogP 5.40
TBAJ-876
(Preclinical)
ClogP 5.15
O
O
O
O
N
N
N
N
H
N
N
2
1
O
(Lead Optimization)
ClogP 1.02
(Lead Optimization)
ClogP 4.39
Fig. 1. Structures of representative Mtb ATP synthase inhibitors.

It was found that the two carbonyls in the cyclobut-3-ene-1,2-dione scaffold
exhibited no interaction with Mtb ATP synthase according to the docking study of
compound 2 [21]. This provided the possibility for scaffold morphing to afford the
furan-2(5H)-ones (3 and 4) (Fig. 2). In addition, maleimide and squaramide fragments
were widely used in a variety of drug molecules as promising pharmacophores [22,
23]. Both fragments generally showed good metabolic stability [24-26]. To reduce the
high in vitro metabolic clearance rate of compound 2, we designed maleimide 5 and
squaramide 6 derived from cyclobut-3-ene-1,2-dione by introducing NH group to
increase the additional hydrogen-bond interaction between the compound and ATP
synthase (Fig. 2).
Fig. 2. Structure modification of compound 2 through scaffold morphing and
increasing H-bond strategy.
Herein, we identified squaramide compound 6ab as a promising anti-TB lead
compound with good anti-TB activity, improved pharmacokinetic and safety profiles
through a preliminary druggability evaluation.
2. Results and discussion
2.1. Chemistry
The target compound 3 was synthesized following the procedure as outlined in
Scheme 1. 2-Ethoxy-2-oxoethyl 2-phenylacetate derivative 7 was treated with
t-BuOK to afford furanone 8 [27]. The sulfonylation of 8 in the presence of
trifluoromethanesulfonic anhydride (Tf2O) and DIPEA led to the intermediate 9 [28].
The replacement of 9 with the corresponding amines via conjugated addition–
elimination in isopropanol resulted in the target compound 3.

Scheme 1. Synthesis of the target compound 3. Reagents and conditions: i) t-BuOK,
THF, argon, rt, 12 h, 52-62%; ii) Tf2O, DIPEA, CH2Cl2, argon, -78 , 2 h, 54-63%;
iii) the corresponding amine, isopropanol, argon, 85 , 3 h, 30-40%.
The synthetic route of compound 4 is shown in Scheme 2. The Suzuki coupling
reaction of 3-amino-4-bromofuran-2(5H)-one 10 and (4-morpholinophenyl)boronic
acid in the presence of Pd(PPh3)4 and Na2CO3 led to the intermediate 11. The
condensation of 11 and the corresponding aldehyde was catalyzed by AcOH under
microwave (MW) condition to afford the intermediate 12. Reduction reaction of 12
with NaBH3CN in 1,2-dichloroethane under argon resulted in the target compound 4.
Scheme 2. Synthesis of the target compound 4. Reagents and conditions: i)
(4-morpholinophenyl)boronic acid, Pd(PPh3)4, Na2CO3, 1,4-dioxane, H2O, argon,
70 , 12 h, 61%; ii) the corresponding aldehyde, AcOH, MeOH, argon, MW, 100
1 h, 57-89%; iii) NaBH3CN, 1,2-dichloroethane, argon, reflux, 6 h, 37-66%.
,
The synthetic route for the target compound 5 is illustrated in Scheme 3. The
heterocyclization of substituted 2-phenylacetamide 13 with diethyl oxalate in the
presence of t-BuOK afforded intermediate 14, which was then converted to vinyl
chloride 15 by oxalyl chloride in CH2Cl2 [29]. The replacement of 15 with the
corresponding amines via addition–elimination under MW condition led to the
formation of target compound 5.

Scheme 3. Synthesis of the target compound 5. Reagents and conditions: i) diethyl
oxalate, t-BuOK, THF, argon, 0 ~ rt, overnight, 93%; ii) oxalyl chloride, DMF,
CH2Cl2, argon, 0 , rt, 4 h, 75%; iii) the corresponding amine, DMF, MW, 150 , 0.5
h, 27-37%.
The preparation of the target compound 6 is described in Scheme 4. Treatment
of 3,4-dimethoxy-3-cyclobutene-1,2-dione 16 with the substituted aniline in ethanol
afforded the intermediate amido-ester 17. The treatment of 17 with the desired
alkylamine in ethanol afforded the target compound 6 via addition–elimination [30].
13
The structures of all new target compounds are characterized by 1H NMR, C NMR
and HRMS.
Scheme 4. Synthesis of the target compound 6. Reagents and conditions: i) the
requisite substituted aniline, ethanol, rt, overnight; ii) the corresponding amine,
ethanol, rt, overnight, 40-94% (two steps).
2.2. Identification of squaramide as a promising lead scaffold
The first effort is to explore the privileged scaffold through the focused
structure-activity relationship (SAR) studies around compound 2 by keeping the
pyridine group on the right-hand side and morpholine or thiomorpholine substituents
on the left. As the change of scaffold may affect the substrate-enzyme interaction, the
position of the nitrogen on pyridine group was adjusted. As shown in Table 1, each

series displayed various in vitro anti-TB activities. Furan-2(5H)-one series (3 and 4)
and maleimide series (5) dramatically lost activity with MIC > 32 μg/mL. We were
pleased to find that squaramide series (6), as exemplified by compounds 6a and 6b,
displayed an MIC of 3.745 μg/mL and 1.544 μg/mL, respectively. The position of the
N atom in the pyridine is also crucial for the activity. For example, compounds with
the 2-pyridinyl substituent could keep the activity (e.g. 6b: MIC = 1.544 μg/mL)
whereas compound 6c lost the anti-TB activity (MIC > 32 μg/mL). In addition, the
substituent on the pyridine ring could affect the activity shown by 6d containing the
3-methyl-2-pyridinyl group with moderate activity while 6e containing
4-methyl-2-pyridinyl group displayed no activity. Subsequently, compounds with an
electron-donating or electron-withdrawing group on the phenyl ring on the left-hand
side were evaluated against TB. Compounds with electron-withdrawing group such as
chloro and trifluoromethyl (6k, 6l) displayed a decreased activity compared to the
compounds with electron-donating groups (6f, 6g, 6h), such as the substituted amino
and methoxy groups. Among compounds 6h, 6i and 6j with a methoxy group at a
different position on the phenyl ring, only compound 6h with the methoxy group at
the 4-position displayed a moderate anti-TB activity with an MIC of 3.417 μg/mL.
The aforementioned SAR result indicated that the rigid four-member ring (squaramide
scaffold) and pyridinyl methyl amino group on the right-hand side played a crucial
role on the anti-TB activity.
Table 1 In vitro anti-TB activities of the target compounds.
MIC1
MIC1
Compds.
R1
Ar
Compds.
R1
Ar
(μg/mL)
(μg/mL)
>32
>32
>32
>32
3a
3c
3b
4a
N

>32
>32
>32
>32
4b
5b
6a
5a
5c
6b
N
N
3.745
1.544
>32
3.646
6c
6d
N
>32
3.758
3.417
>32
6e
6g
6i
6f
6h
6j
1.996
6.024
4-CH3O
2-CH3O
CF3
3-CH3O
Cl
7.698
0.122
14.896
0.038
6k
2
6l
INH
1 Minimum inhibitory concentration against M. tuberculosis H37Rv.
To further understand the SAR of squaramide series (6), structural modification
on the left-hand side was explored by introducing substituted aromatic rings or
heteroaryl rings. As displayed in Table 2, compounds (6m, 6n) with pyridinyl instead
of phenyl lost activity (MIC > 32 μg/mL). Expanding morpholine to 1,4-oxazepane
(6o, MIC 6.649 μg/mL) or inserting carbonyl on the morpholine ring (6p, MIC 7.751
μg/mL) led to a weaker activity compared to 6a. Sulfoxide 6q had a much lower
activity compared to its parent 6b. Compound 6r with a meta-substituent had no
activity (MIC > 32 μg/mL) while the para-substituent was beneficial (6b). It was
noted that compounds with two substituents at the ortho-position exhibited an
improved activity compared to the compounds with mono-substituent, exemplified by
6s (MIC 2.452 μg/mL) vs 6a (MIC 3.745 μg/mL), and 6t (MIC 0.496 μg/mL) vs 6b
(MIC 1.544 μg/mL), respectively. Moreover, compared to compound 6t, compounds
6u, 6v and 6w with substituents such as cyano, trifluoromethyl and methoxy,
respectively, displayed a reduced activity. Compound 6w with the methoxy
ortho-substitution totally lost activity.
With this critical SAR between the substituent size, position and anti-TB activity

in hand, a series novel squaramide with benzo aliphatic ring (6aa, 6ae, 6af, 6ag) or
benzo heterocyclic ring (6ab, 6ac, 6ad, 6ah, 6ai, 6aj and 6ak) on the left-hand side
were further investigated. We were pleased to find that all compounds exhibited good
anti-TB activity with MICs ranging from 0.452 ~ 3.456 μg/mL, except compounds
containing benzothiophene (6aj) or benzopyrrole (6ak). Particularly, these active
compounds showed no cytotoxicity against Vero cell (IC50 > 64 μg/mL) and low
lipophilicity with ClogP ranging from 0.84 ~ 1.81, indicating these compounds may
display favorable pharmacokinetic properties.
Table 2 The MIC, IC50 and ClogP values of squaramide compounds
MIC2
IC50(Vero)
Compds.
R
ClogP1
(µg/mL)
(μg/mL)
0.25
1.08
0.27
0.27
0.30
3.745
6a
6b
6m
6n
6o
>64
1.544
>32
>64
ND3
>32
ND
ND
6.649
0.39
7.751
ND
6p
-0.63
1.08
0.75
1.58
1.33
15.828
>32
ND
ND
>64
>64
>64
6q
6r
6s
6t
2.452
0.496
1.232
6u

2.58
1.21
1.81
9.402
>32
ND
ND
>64
6v
6w
6aa
0.979
0.89
0.84
1.00
0.92
0.92
0.452
1.885
2.222
3.456
1.832
>64
>64
>64
>64
>64
6ab
6ac
6ad
6ae
6af
1.33
1.52
1.52
1.98
0.739
0.963
1.909
>64
>64
>64
ND
6ag
6ah
6ai
>32
>32
6aj
0.78
1.02
ND
>64
6ak
0.122
0.038
2
INH
1
2
Calculated with ChemDraw Professional 16.0. Minimum inhibitory concentration against
M. tuberculosis H37Rv. 3 ND, not determined.
2.3. Evaluation of compound 6ab as a potential lead for treatment of TB
Squaramide compounds with good in vitro activities (MIC < 1 μg/mL) were
selected for further tests against two MDR (XDR)-TB clinical isolated strains (Table
3). All compounds demonstrated potent activities against drug-resistant TB strains
with MIC < 1 μg/mL. The results indicated that this scaffold has the potential to treat

drug-resistant tuberculosis.
Table 3 Activity of compounds against the selected clinical isolates of M.
tuberculosis.
MIC (μg/mL)
Compds.
H37Rv1
139462
148623
2
0.120
0.748
1.753
0.483
0.962
3.634
0.019
0.015
0.107
0.257
0.903
0.470
0.488
3.531
2.264
>10
0.114
0.207
0.940
0.499
0.895
3.374
>10
6t
6aa
6ab
6ag
6ah
INH
RFP
9.317
1
Retested. 2 Resistance to isoniazid (INH), streptomycin (SM), rifampicin (RFP), ethambutol
(EMB), rifabutin (RBT), paza-aminosalicylate (PAS) and ofloxacin (OLFX). 3 Resistance to INH,
SM, RFP, EMB, PAS, prothionamide and capreomycin (CPM).
Due to the poor pharmacokinetic profile of the lead compound 2 [21], some
representative squaramide compounds were chosen for the investigation of metabolic
stability. As shown in Table 4, compound 6a exhibited significantly improved
stability both in mouse and human hepatocytes with much longer half-life (t1/2 > 93.2
min) and lower intrinsic clearance (Clint < 7.4 µL/min/million cell), compared to
compound 2. As such, the design strategy of bringing more polarity appeared to
improve the metabolic stability. The poor metabolic stability of compounds 6b and 6t
may be attributed to the oxidation of sulfur. Compound 6aa was unstable in both
mouse and human hepatocytes. Compounds 6ag and 6ab, showed good to excellent
stability in human hepatocytes, compared to compound 2. The above results
encouraged us to further explore the safety profiles of this squaramide series.

Table 4 Mouse and human hepatocytes stability of selected compounds.
t1/2
Clint
(min)
(µL/min/million cell)
Compds.
Mouse Human Mouse
Human
21.9
<7.4
57.2
77.0
67.1
<7.4
16.6
2
17.1
31.7
40.6
<7.4
55.1
127.8
62.4
40.8
67.2
6a
>93.2 >93.2
6b
12.6
5.4
12.1
9.0
6t
6aa
6ab
6ag
11.1
17.0
10.3
10.3
>93.2
41.9
Subsequently, selected compounds with good anti-TB activity and metabolic
stability were screened for hERG inhibitory activity (Table 5). The low inhibition of
hERG (IC50 > 10 μM) indicated that squaramides 6 may display a low risk of
blocking the cardiac potassium channel which may cause the QT prolongation.
Table 5 The hERG inhibition of squaramide compounds
hERG
IC50 (μM)
>30
hERG
IC50 (μM)
>30
Compds.
Compds.
2
6aa
6ab
6a
6b
6t
>30
>30
>30
6ah
Dofetilide1
>10
>10
0.013
1 Positive control.
To further evaluate the druggability of compound 6ab, the pharmacokinetic
studies in BALB/c mice was conducted, following a single oral or an intravenous
administration (Table 6). Compound 6ab displayed a good PK profile as reflected by
a high plasma exposure (AUC0−∞ = 2198 ng h/mL) and a high maximal plasma
concentration (Cmax = 1333 ng/mL) after an oral administration at 50 mg/kg. In
addition, compound 6ab had a moderate clearance with CL of 51.4 mL/min/kg after
an intravenously administration at a dose of 5 mg/kg. Thus far, all the above results
supported compound 6ab to be further evaluated as a promising potential candidate
for the DR-TB treatment.

Table 6 Mouse pharmacokinetic parameters of compound 6ab
1
F4
2
3
dose
Cmax
tmax t1/2
AUC0-t
Clearance
AUC0-∞ MRT0-∞
(mg/kg) (ng/mL) (h)
(h) (ng·h/mL) (ng·h/mL)
(h)
(mL/min/kg) (%)
po
iv
50
5
1333 0.83 0.51
0.33
2197
1667
2198
1672
1.36
0.28
13.1
51.4
1 Plasma elimination half-life. 2 Plasma exposure. 3 Mean residence time. 4 Oral bioavailability.
2.4. Docking studies with 6ab as a potential ATP synthase inhibitor
In order to investigate the binding mode of the squaramide in the active site of
Mtb ATP synthase, the most promising compound 6ab was docked into the active site
of the enzyme using the Discovery Studio 2018 according to the reported homology
model of Mtb ATP synthase and the receptor cavity (active site of the protein) [21].
The conformation corresponding to the highest docking score with CDOCKER
protocol was selected as the best binding pose. As exhibited in Fig. 3, the pyridine
moiety of compound 6ab formed a hydrogen bond interaction with Arg186, which is
consistent with the proposed binding mode of the pyridine in compound 2.
Furthermore, the introduction of nitrogen atom formed a key hydrogen bond with
Gly62, which could provide an additional binding affinity. In addition, the benzene
moiety formed a Pi-anion interaction with Glu65, which plays a crucial role in the
interaction of bedaquiline with Mtb ATP synthase [15]. The dioxolane moiety could
form a water hydrogen bond as compound 2. Compound 6ab could also form the
non-conventional interaction with Ala66, Leu183, Ile59 and Ser182 to improve the
affinity.

Fig. 3. Predicted binding mode of compound 6ab with Mtb ATP synthase.
To further confirm the anti-TB mechanism of squaramides, we measured the
activities of the selected squaramides against a bedaquiline-resistant strain. As shown
in Table 7, compounds 6t and 6ab showed potent activities against the drug-sensitive
H37Rv strain as well as compound 2 and bedaquiline, but they all demonstrated
decreased potency against the bedaquiline-resistant strain. The MIC fold shifts of
compounds 6t, 6ab, 2 and bedaquiline were 5.7, 7.5, 14.9 and 62, respectively. In this
assay, the known drug INH showed the same sensitivity to H37Rv and the
bedaquiline-resistant strain. Combined with the result that compounds 6t and 6ab
displayed potent activity against two MDR (XDR)-TB clinical isolated strains
(excluding the resistance strain to bedaquline) as compound 2, an inhibitor of the Mtb
ATP synthase. It suggested that squaramides 6t, 6ab may exert anti-TB activity
through the inhibition of Mtb ATP synthase.
Table 7 Activity of selected compounds against bedaquiline-resistant strain
MIC (μg/mL)
H37Rv1 resistant strain2
MIC
Foldshift3
14.9
5.7
Compds.
2
0.202
0.845
0.500
3.016
4.851
3.758
2.235
0.052
6t
6ab
7.5
Bedaquiline 0.036
INH 0.038
62
-
1 Retested. 2 Resistance to bedaquiline.3 Ratio of MIC values against bedaquiline-resistant strain
and H37Rv.
3. Conclusion
In summary, we have identified a class of novel squaramide derivatives (6) as
anti-TB
agents
through
hydrogen-bond
increasing
strategy
from
cyclobut-3-ene-1,2-diones compound 2. Compound 6ab exhibited potent in vitro
activity against H37Rv as well as two MDR (XDR)-TB clinical isolated strains.
Preliminary druggability evaluation of compound 6ab displayed good metabolic
stability, low cytotoxicity, and acceptable oral bioavailability. The docking study
revealed the key interactions of compound 6ab with Arg186, Glu65 and Gly62

residues of Mtb ATP synthase, similar to compound 2. The result of in vitro
anti-bedaquiline-resistant strain assay further supported that compound 6ab may
exhibit anti-TB activity via the inhibition of ATP synthase. Hence, compound 6ab is a
promising anti-TB lead compound warranting further optimization, which will be
reported in due course.
4. Experimental
4.1. General experimental information
All the solvents and chemicals were obtained from commercial sources and used
without further purification. TLC was performed on silica gel plates (GF254) with
visualization of components by UV light (254 nm). Column chromatography was
carried out on silica gel (200-300 mesh). The structural identities of the prepared
compounds were confirmed by 1H NMR and 13C NMR spectroscopy and
1
high-resolution mass spectrometry (HRMS). H NMR spectra were obtained on
Varian Mercury-400 at 400 MHz or Varian Mercury-500 at 500 MHz. 13C NMR
spectra were obtained on Bruker-400 at 100 MHz. Chemical shifts values were
referenced to the residual solvent peak and reported in ppm (δ scale) and all coupling
constant (J) values were given in Hz. CDCl3 or DMSO d6 were used as the standard
NMR solvents. The following multiplicity abbreviations are used: (s) singlet, (d)
doublet, (t) triplet, (q) quartet, (m) multiplet and (br) broad. ESI-HRMS data were
measured on Thermo Exactive Orbitrap plus spectrometer. Melting points were
determined on Yanaco MP-J3 microscope melting point apparatus.
4.2. Experimental procedures
4.2.1. General procedure for the synthesis of compound 8
To a magnetically stirred solution of intermediate 7 (5.0 mmol, 1.0 equiv.) in dry
THF (50 mL) was added t-BuOK (10.0 mmol, 2.0 equiv.). The reaction mixture was
stirred under argon atmosphere at room temperature for 12 h. The reaction was
quenched with 10 mL H2O and the pH was adjusted to 1 with HCl (1N). The solvent
was concentrated under reduced pressure, the residue was purified by column
chromatography (1~3 % MeOH/CH2Cl2) to afford compound 8.
1
4.2.1.1. 4-Hydroxy-3-(4-morpholinophenyl)furan-2(5H)-one (8a). Yield 61.6%. H

NMR (500 MHz, DMSO d6) δ 12.44 (s, 1H), 7.79 (d, J = 8.5 Hz, 2H), 6.95 (d, J =
8.6 Hz, 2H), 4.73 (s, 2H), 3.73 (d, J = 4.0 Hz, 4H), 3.10 (s, 4H).
4.2.1.2. 4-Hydroxy-3-(4-thiomorpholinophenyl)furan-2(5H)-one (8b). Yield 51.5%.
1H NMR (400 MHz, DMSO d6) δ 8.12 (d, J = 8.8 Hz, 2H), 6.74 (d, J = 8.8 Hz, 2H),
3.94 (s, 2H), 3.35 – 3.30 (m, 4H), 2.72 – 2.66 (m, 4H).
4.2.2 General procedure for the synthesis of compound 9
DIPEA (1.5 mmol, 1.5 equiv.) was added to a stirred solution of compound 8
(1.0 mmol, 1.0 equiv.) in dry CH2Cl2 (20 mL) at room temperature. The solution was
cooled to -78 °C and Tf2O (1.2 mmol, 1.2 equiv.) in 10 mL dry CH2Cl2 was slowly
added under argon atmosphere. The reaction mixture was slowly risen to 0 °C and
stirred for additional 2 h. The reaction mixture was diluted with CH2Cl2 (20 mL) and
washed with NaHCO3 (20 mL), brine (20 mL), dried over Na2SO4. The filtrate was
removed under reduced pressure, the residue was purified by column chromatography
(1% MeOH/CH2Cl2) to afford compound 9.
4.2.2.1.
4-(4-Morpholinophenyl)-5-oxo-2,5-dihydrofuran-3-yl
1
trifluoromethanesulfonate (9a). Yield 62.6%. H NMR (400 MHz, CDCl3) δ 7.78 (d,
J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 5.05 (s, 2H), 3.96 – 3.80 (m, 4H), 3.34 –
3.19 (m, 4H).
4.2.2.2.
4-(4-Thiomorpholinophenyl)-5-oxo-2,5-dihydrofuran-3-yl
1
trifluoromethanesulfonate (9b). Yield 53.9%. H NMR (400 MHz, CDCl3) δ 7.87 –
7.70 (m, 2H), 6.96 (d, J = 8.9 Hz, 2H), 5.04 (s, 2H), 3.76 – 3.68 (m, 4H), 2.79 – 2.72
(m, 4H).
4.2.3. General procedure for the synthesis of the target compound 3
A capped tube was charged with compound 9 (0.2 mmol, 1.0 equiv.) and
corresponding amine (0.4 mmol, 2.0 equiv.) in isopropanol (5 mL). The tube was
flushed with argon, capped, and heated at 85 ℃ for 3 h. After cooling to room
temperature, the solvent was removed under reduced pressure, the residue was
purified by column chromatography (2% MeOH/CH2Cl2) to afford the target
compound 3.
4.2.3.1. 3-(4-Morpholinophenyl)-4-((pyridin-2-ylmethyl)amino)furan-2(5H)-one (3a).

Brown solid, yield 34.1%. mp 159-161 °C. 1H NMR (500 MHz, CDCl3) δ 8.58 (d, J =
3.5 Hz, 1H), 7.70 (s, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 7.6 Hz, 1H), 6.98 (d, J
= 8.1 Hz, 2H), 6.49 (s, 1H), 4.83 (s, 2H), 4.44 (d, J = 4.9 Hz, 2H), 3.87 (brs, 4H), 3.18
13
(brs, 4H). C NMR (100 MHz, CDCl3) δ 174.1, 161.1, 155.0, 149.9, 149.5, 137.1,
128.6, 123.0, 122.5, 121.4, 116.0, 96.0, 66.9, 65.4, 49.2, 48.1. HRMS (ESI): m/z
[M+H]+ calcd for C20H22N3O3, 352.1656; found, 352.1652.
4.2.3.2. 3-(4-Morpholinophenyl)-4-((pyridin-3-ylmethyl)amino)furan-2(5H)-one (3b).
1
Brown solid, yield 39.8%. mp 246-248 °C. H NMR (500 MHz, CDCl3) δ 8.57 (s,
1H), 8.54 (s, 1H), 7.61 (d, J = 7.3 Hz, 1H), 7.39 – 7.30 (m, 3H), 6.93 (d, J = 8.3 Hz,
2H), 5.59 (brs, 1H), 4.76 (s, 2H), 4.36 (d, J = 5.7 Hz, 2H), 3.85 (s, 4H), 3.15 (s, 4H).
13C NMR (100 MHz, CDCl3) δ 173.8, 161.0, 150.2, 149.6, 148.5, 134.9, 132.6, 128.8,
124.0, 121.7, 116.0, 97.0, 66.8, 65.2, 49.1, 45.6. HRMS (ESI): m/z [M+H]+ calcd for
C20H22N3O3, 352.1656; found, 352.1650.
4.2.3.3. 3-(4-Thiomorpholinophenyl)-4-((pyridin-2-ylmethyl)amino)furan-2(5H)-one
1
(3c). Brown solid, yield 29.5%. mp 146-148 °C. H NMR (400 MHz, CDCl3) δ 8.58
(d, J = 4.6 Hz, 1H), 7.71 (td, J = 7.7, 1.7 Hz, 1H), 7.47 (d, J = 8.7 Hz, 2H), 7.28 –
7.24 (m, 1H), 7.22 (d, J = 7.8 Hz, 1H), 6.98 (d, J = 7.4 Hz, 2H), 6.51 (brs, 1H), 4.84
13
(s, 2H), 4.44 (d, J = 5.4 Hz, 2H), 3.60 – 3.56 (m, 4H), 2.76 (brs, 4H). C NMR (100
MHz, CDCl3) δ 174.1, 161.2, 155.0, 149.5, 137.1, 128.7, 123.0, 121.5, 117.4, 95.8,
65.4, 52.1, 48.1, 26.6. HRMS (ESI): m/z [M+H]+ calcd for C20H22N3O2S, 368.1427;
found, 368.1426.
4.2.4. Synthesis of 3-amino-4-(4-morpholinophenyl)furan-2(5H)-one (11a)
To a mixture of 3-amino-4-bromofuran-2(5H)-one 10 (1.0 mmol, 1.0 equiv.),
(4-morpholinophenyl)boronic acid (1.2 mmol, 1.2 equiv.) and Na2CO3 (1.2 mmol, 1.2
equiv.) in dioxane (5 mL) and H2O (1 mL) was added Pd(PPh3)4 (0.05 mmol, 0.05
equiv.). The mixture was degassed and stirred at 70 °C under argon atmosphere for 12
h. The reaction mixture was cooled to room temperature, diluted with CH2Cl2 (20
mL), dried over Na2SO4 and filtrated. The solvent was removed under reduced
pressure, the residue was purified by column chromatography (1% MeOH/CH2Cl2) to
afford compound 11a. Yellow solid, Yield 61.0%. 1H NMR (500 MHz, CDCl3) δ 7.32

(d, J = 8.3 Hz, 2H), 6.97 (d, J = 8.3 Hz, 2H), 5.00 (s, 2H), 3.94 (brs, 2H), 3.92 – 3.82
(m, 4H), 3.30 – 3.16 (m, 4H).
4.2.5. General procedure for the synthesis of compound 12
To a solution of compound 11 (0.2 mmol, 1.0 equiv.) and the corresponding
aldehyde (0.3 mmol, 1.5 equiv.) in dry MeOH (5 mL) was added catalytic amount of
acetic acid (2 µL). The mixture was heated in MW at 100 °C under argon atmosphere
for 1 h. After cooling to room temperature, the precipitate was filtered and washed
with methanol to afford compound 12.
4.2.5.1. 4-(4-Morpholinophenyl)-3-((pyridin-2-ylmethylene)amino)furan-2(5H)-one
(12a). Yellow solid, yield 57.2%. 1H NMR (500 MHz, CDCl3) δ 9.44 (s, 1H), 8.75 (d,
J = 3.3 Hz, 1H), 8.20 (d, J = 7.7 Hz, 1H), 7.87 (d, J = 8.4 Hz, 2H), 7.82 (t, J = 7.6 Hz,
1H), 7.42 – 7.34 (m, 1H), 6.95 (d, J = 8.4 Hz, 2H), 5.21 (s, 2H), 3.87 (brs, 4H), 3.30
(brs, 4H).
4.2.5.2. 4-(4-Morpholinophenyl)-3-((pyridin-3-ylmethylene)amino)furan-2(5H)-one
(12b). Yellow solid, yield 88.7%. 1H NMR (400 MHz, CDCl3) δ 9.52 (s, 1H), 9.17 (s,
1H), 8.72 (d, J = 4.2 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 8.7 Hz, 2H), 7.46
(dd, J = 7.7, 4.8 Hz, 1H), 6.96 (d, J = 8.8 Hz, 2H), 5.23 (s, 2H), 3.90 – 3.86 (m, 4H),
3.34 – 3.30 (m, 4H).
4.2.6. General procedure for the synthesis of the target compound 4
To a solution of compound 12 (0.1 mmol, 1.0 equiv.) in dry 1,2-dichloroethane
(5 mL) was added NaBH3CN (0.11 mmol, 1.1 equiv.). The mixture was refluxed
under argon atmosphere for 6 h. The solvent was removed under reduced pressure, the
residue was purified by column chromatography (1~3% MeOH/CH2Cl2) to afford the
target compound 4.
4.2.6.1. 4-(4-Morpholinophenyl)-3-((pyridin-2-ylmethyl)amino)furan-2(5H)-one (4a).
1
Yellow solid, yield 65.5%. mp 73-75 °C. H NMR (400 MHz, CDCl3) δ 8.54 (d, J =
4.4 Hz, 1H), 7.62 (td, J = 7.7, 1.6 Hz, 1H), 7.33 (d, J = 8.7 Hz, 2H), 7.20 – 7.13 (m,
2H), 6.92 (d, J = 8.8 Hz, 2H), 5.05 (brs, 1H), 4.93 (s, 2H), 4.44 (s, 2H), 3.93 – 3.82
(m, 4H), 3.26 – 3.18 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 172.0, 157.8, 150.9,
149.2, 136.7, 128.3, 128.1, 125.9, 122.8, 122.2, 121.6, 115.0, 69.8, 66.7, 49.6, 48.5.

HRMS (ESI): m/z [M+H]+ calcd for C20H22N3O3, 352.1656; found, 352.1643.
4.2.6.2. 4-(4-Morpholinophenyl)-3-((pyridin-3-ylmethyl)amino)furan-2(5H)-one (4b).
Yellow solid, yield 37.0%. mp 85-87 °C. 1H NMR (500 MHz, CDCl3) δ 8.50 (s, 1H),
8.32 (s, 1H), 7.56 (d, J = 7.5 Hz, 1H), 7.32 – 7.18 (m, 3H), 6.92 (d, J = 8.4 Hz, 2H),
4.91 (s, 2H), 4.29 (brs, 3H), 3.96 – 3.76 (m, 4H), 3.34 – 3.09 (m, 4H). 13C NMR (100
MHz, CDCl3) δ 172.0, 151.2, 148.7, 148.4, 135.5, 134.8, 128.4, 127.5, 127.3, 123.7,
122.3, 114.9, 70.0, 66.7, 48.3, 45.9. HRMS (ESI): m/z [M+H]+ calcd for C20H22N3O3,
352.1656; found, 352.1644.
4.2.7. Synthesis of 3-hydroxy-4-(4-morpholinophenyl)-1H-pyrrole-2,5-dione (14a)
To a solution of compound 13 (10.0 mmol, 1.0 equiv.) and diethyl oxalate (15.0
mmol, 1.5 equiv.) in dry DMF (50 mL) was added t-BuOK (30.0 mmol, 3.0 equiv.) in
portion at 0 , the reaction mixture was stirred at room temperature under argon
atmosphere overnight. The reaction mixture was diluted with water (100 mL) and
acidified with 6N HCl to pH = 1. The precipitate was filtered, washed with H2O and
1
dried to afford compound 14a. Yellow solid, yield 93.0%. H NMR (400 MHz,
DMSO d6) δ 9.65 (brs, 1H), 8.02 (s, 2H), 6.86 (s, 2H), 3.82 – 3.64 (m, 4H), 3.13 –
2.94 (m, 4H).
4.2.8. Synthesis of 3-chloro-4-(4-morpholinophenyl)-1H-pyrrole-2,5-dione (15a)
A solution of oxalyl chloride (7.7 mmol, 1.1 equiv.) in dry CH2Cl2 (10 mL) was
added dropwise to a solution of intermediate 14a (7.0 mmol, 1.0 equiv.) in dry DMF
(5 mL) and CH2Cl2 (5 mL) at 0 . After stirring at room temperature for 4 h, the
reaction mixture was poured into cold water (50 mL) and extracted with CH2Cl2 (50
mL), washed with NaHCO3, brine, and dried over Na2SO4. The solvent was removed
under reduced pressure and the residue was purified by column chromatography
(1~20% ethyl acetate/petroleum) to afford the compound 15a. Yellow solid, yield
1
75.2%. H NMR (400 MHz, DMSO d6) δ 11.33 (brs, 1H), 7.88 (d, J = 9.1 Hz, 2H),
7.07 (d, J = 9.2 Hz, 2H), 3.77 – 3.71 (m, 4H), 3.30 – 3.25 (m, 4H).
4.2.9. General procedure for the synthesis of the target compound 5
To a solution of compound 15a (0.2 mmol, 1.0 equiv.) in DMF (1 mL) was

added corresponding amine (0.4 mmol, 2.0 equiv.). The reaction mixture was heated
in MW at 150 °C for 0.5 h. The solvent was removed under reduced pressure and the
residue was purified by column chromatography (1~3 %MeOH/CH2Cl2) to afford the
target compound 5.
4.2.9.1. 3-(4-Morpholinophenyl)-4-((pyridin-2-ylmethyl)amino)-1H-pyrrole-2,5-dione
1
(5a). Yellow solid, yield 33.8%. mp 231-233 . H NMR (400 MHz, CDCl3) δ 8.57
(d, J = 4.3 Hz, 1H), 7.65 (td, J = 7.7, 1.7 Hz, 1H), 7.31 (d, J = 8.7 Hz, 2H), 7.25 –
7.17 (m, 2H), 7.05 (d, J = 7.9 Hz, 1H), 6.95 (d, J = 8.5 Hz, 2H), 6.74 (brs, 1H), 4.47
13
(d, J = 5.2 Hz, 2H), 3.92 – 3.86 (m, 4H), 3.24 – 3.19 (m, 4H). C NMR (100 MHz,
CDCl3) δ 171.9, 167.7, 155.0, 150.5, 149.2, 141.7, 136.7, 131.0, 122.7, 121.7, 120.8,
115.1, 102.0, 66.9, 49.0, 48.2. HRMS (ESI): m/z [M+H]+ calcd for C20H21N4O3,
365.1608; found, 365.1596.
4.2.9.2. 3-(4-Morpholinophenyl)-4-((pyridin-3-ylmethyl)amino)-1H-pyrrole-2,5-dione
1
(5b). Yellow solid, yield 27.4%. mp 219-221 °C. H NMR (500 MHz, CDCl3) δ 8.53
(brs, 1H), 8.30 (s, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.33 (s, 1H), 7.26 – 7.21 (m, 1H),
7.18 (d, J = 8.3 Hz, 2H), 6.89 (d, J = 8.3 Hz, 2H), 5.57 (brs, 1H), 4.39 (d, J = 5.8 Hz,
2H), 3.89 – 3.83 (m, 4H), 3.22 – 3.16 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 171.5,
167.6, 150.8, 149.3, 149.0, 140.9, 135.4, 135.1, 131.2, 123.6, 120.0, 115.0, 103.1,
66.8, 48.8, 45.4. HRMS (ESI): m/z [M+H]+ calcd for C20H21N4O3, 365.1608; found,
365.1596.
4.2.9.3. 3-(4-Morpholinophenyl)-4-((pyridin-4-ylmethyl)amino)-1H-pyrrole-2,5-dione
1
(5c). Yellow solid, yield 37.0%. mp 245-247 °C. H NMR (500 MHz, CDCl3) δ 8.55
(d, J = 5.2 Hz, 2H), 7.68 (brs, 1H), 7.50 (d, J = 8.7 Hz, 2H), 7.21 (d, J = 4.2 Hz, 2H),
13
6.91 (d, J = 8.7 Hz, 2H), 3.98 (s, 2H), 3.89 – 3.83 (m, 4H), 3.29 – 3.24 (m, 4H). C
NMR (100 MHz, CDCl3) δ 171.3, 170.7, 152.3, 149.3, 147.6, 140.2, 132.8, 130.9,
123.9, 118.6, 114.4, 66.6, 47.7, 29.4. HRMS (ESI): m/z [M+H]+ calcd for C20H21N4O3,
365.1608; found, 365.1593.
4.2.10. General procedure for the synthesis of the target compound 6
To a solution of 3,4-dimethoxy-3-cyclobutene-1,2-dione 16 (1.0 mmol, 1.0

equiv.) in ethanol (10 mL) was added substituted aniline (1.0 mmol, 1.0 equiv.). The
reaction mixture was stirred at room temperature overnight. The precipitate was
filtered, washed with cooled ethanol and dried to afford the intermediate 17. To
suspended solution of 17 in ethanol (10 mL) was added the corresponding amines (1.2
mmol, 1.2 equiv.). The reaction mixture was stirred at room temperature overnight.
The precipitate was filtered, wash with cooled ethanol and dried to afford the target
compound 6.
4.2.10.1.
3-((4-Morpholinophenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,2-dio
1
ne (6a). Yellow solid, yield 89.3% (two steps). mp > 250 . H NMR (400 MHz,
DMSO d6) δ 9.70 (brs, 1H), 8.60 (dd, J = 4.8, 1.6 Hz, 1H), 8.09 (brs, 1H), 7.83 (td, J
= 7.7, 1.8 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.36 (dd, J = 6.1, 1.3 Hz, 1H), 7.32 (d, J
= 9.2 Hz, 2H), 6.94 (d, J = 9.0 Hz, 2H), 4.92 (d, J = 5.7 Hz, 2H), 3.77 – 3.70 (m, 4H),
13
3.08 – 3.02 (m, 4H). C NMR (100 MHz, DMSO d6) δ 183.2, 180.7, 168.5, 163.7,
157.0, 149.1, 147.0, 137.1, 131.1, 122.6, 121.6, 119.2, 116.0, 66.0, 48.8, 48.3. HRMS
(ESI): m/z [M+H]+ calcd for C20H21N4O3, 365.1608; found, 365.1604.
4.2.10.2.
3-((Pyridin-2-ylmethyl)amino)-4-((4-thiomorpholinophenyl)amino)cyclobut-3-ene-1,2
1
-dione (6b). Off-white solid, yield 48.9% (two steps). mp > 250 °C. H NMR (400
MHz, DMSO d6) δ 9.69 (brs, 1H), 8.60 (ddd, J = 4.9, 1.7, 0.9 Hz, 1H), 8.08 (brs,
1H), 7.83 (td, J = 7.7, 1.8 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.35 (ddd, J = 7.5, 4.8,
1.0 Hz, 1H), 7.31 (d, J = 8.9 Hz, 2H), 6.92 (d, J = 9.0 Hz, 2H), 4.92 (d, J = 5.5 Hz,
13
2H), 3.46 – 3.41 (m, 4H), 2.69 – 2.64 (m, 4H). C NMR (100 MHz, DMSO d6) δ
183.2, 180.7, 168.5, 163.7, 157.0, 149.1, 146.8, 137.1, 131.0, 122.7, 121.6, 119.3,
117.4, 51.5, 48.3, 25.6. HRMS (ESI): m/z [M+H]+ calcd for C20H21N4O2S, 381.1380;
found, 381.1372.
4.2.10.3.
3-((Pyridin-3-ylmethyl)amino)-4-((4-thiomorpholinophenyl)amino)cyclobut-3-ene-1,2
-dione (6c). White solid, yield 93.7% (two steps). mp > 250 ℃. 1H NMR (400 MHz,

DMSO d6) δ 9.53 (brs, 1H), 8.61 (d, J = 1.7 Hz, 1H), 8.53 (dd, J = 4.8, 1.7 Hz, 1H),
7.88 (brs, 1H), 7.82 – 7.78 (m, 1H), 7.42 (ddd, J = 7.9, 4.8, 0.8 Hz, 1H), 7.27 (d, J =
8.9 Hz, 2H), 6.91 (d, J = 9.1 Hz, 2H), 4.84 (d, J = 6.0 Hz, 2H), 3.46 – 3.41 (m, 4H),
13
2.68 – 2.64 (m, 4H). C NMR (100 MHz, DMSO d6) δ 183.2, 180.7, 168.2, 163.9,
148.9, 148.7, 146.8, 135.4, 134.2, 130.8, 123.7, 119.5, 117.3, 51.4, 44.6, 25.6. HRMS
(ESI): m/z [M+H]+ calcd for C20H21N4O2S, 381.1380; found, 381.1377.
4.2.10.4.
3-(((6-Methylpyridin-2-yl)methyl)amino)-4-((4-thiomorpholinophenyl)amino)cyclobut
1
-3-ene-1,2-dione (6d). Yellow solid, yield 65.1% (two steps). mp > 250 °C. H NMR
(400 MHz, DMSO d6) δ 9.66 (brs, 1H), 8.01 (brs, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.30
(d, J = 8.9 Hz, 2H), 7.20 (d, J = 7.7 Hz, 2H), 6.92 (d, J = 9.0 Hz, 2H), 4.85 (d, J = 4.9
Hz, 2H), 3.46 – 3.42 (m, 4H), 2.69 – 2.64 (m, 4H), 2.49 (s, 3H). 13C NMR (100 MHz,
DMSO d6) δ 183.2, 180.6, 168.4, 163.7, 157.7, 156.5, 146.8, 137.4, 130.9, 122.0,
119.4, 118.5, 117.4, 51.5, 48.5, 25.6, 23.9. HRMS (ESI): m/z [M+H]+ calcd for
C21H23N4O2S, 395.1536; found, 395.1531.
4.2.10.5.
3-(((5-Methylpyridin-2-yl)methyl)amino)-4-((4-thiomorpholinophenyl)amino)cyclobut
1
-3-ene-1,2-dione (6e). Off-white solid, yield 44.8% (two steps). mp > 250 °C. H
NMR (500 MHz, CDCl3) δ 8.36 (s, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.45 – 7.36 (m, 1H),
7.31 (d, J = 7.8 Hz, 2H), 6.91 (d, J = 7.0 Hz, 2H), 4.88 (s, 2H), 3.54 – 3.41 (m, 4H),
2.83 – 2.70 (m, 4H), 2.37 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 183.7, 181.8, 168.6,
164.8, 153.8, 149.4, 148.5, 138.8, 133.3, 131.5, 122.9, 120.5, 118.7, 52.8, 48.8, 27.0,
18.2. HRMS (ESI): m/z [M+H]+ calcd for C21H23N4O2S, 395.1536; found, 395.1538.
4.2.10.6.
3-((4-(Piperidin-1-yl)phenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,2
1
-dione (6f). Off-white solid, yield 55.2% (two steps). mp > 250 °C. H NMR (500
MHz, DMSO d6) δ 9.66 (brs, 1H), 8.59 (s, 1H), 8.05 (brs, 1H), 7.83 (t, J = 7.7 Hz,
1H), 7.42 (d, J = 7.2 Hz, 1H), 7.35 (s, 1H), 7.28 (d, J = 7.2 Hz, 2H), 6.91 (d, J = 8.0
13
Hz, 2H), 4.92 (s, 2H), 3.06 (s, 4H), 1.61 (s, 4H), 1.51 (s, 2H). C NMR (100 MHz,

DMSO d6) δ 183.1, 180.7, 168.5, 163.7, 157.0, 149.1, 147.8, 137.1, 130.5, 122.7,
121.6, 119.2, 116.7, 50.0, 48.3, 25.2, 23.7. HRMS (ESI): m/z [M+H]+ calcd for
C21H23N4O2, 363.1816; found, 363.1808.
4.2.10.7.
3-((4-(Dimethylamino)phenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,
1
2-dione (6g). Yellow solid, yield 46.7% (two steps). mp 248-250 °C. H NMR (400
MHz, DMSO d6) δ 9.63 (brs, 1H), 8.61 – 8.58 (m, 1H), 8.03 (brs, 1H), 7.83 (td, J =
7.7, 1.7 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.35 (dd, J = 7.3, 5.0 Hz, 1H), 7.27 (d, J =
8.8 Hz, 2H), 6.73 (d, J = 9.0 Hz, 2H), 4.92 (d, J = 5.5 Hz, 2H), 2.85 (s, 6H). 13C NMR
(100 MHz, DMSO d6) δ 182.9, 180.8, 168.3, 163.7, 157.1, 149.1, 146.9, 137.1,
128.7, 122.6, 121.6, 119.5, 113.1, 48.3, 40.4. HRMS (ESI): m/z [M+H]+ calcd for
C18H19N4O2, 323.1503; found, 323.1497.
4.2.10.8.
3-((4-Methoxyphenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,2-dione
1
(6h). Off-white solid, yield 61.3% (two steps). mp 206-208 . H NMR (500 MHz,
DMSO d6) δ 9.72 (brs, 1H), 8.60 (d, J = 4.0 Hz, 1H), 8.08 (brs, 1H), 7.83 (t, J = 7.5
Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.39 – 7.33 (m, 3H), 6.92 (d, J = 8.5 Hz, 2H), 4.92
(s, 2H), 3.73 (s, 3H). 13C NMR (100 MHz, DMSO d6) δ 183.4, 180.6, 168.6, 163.7,
157.0, 155.2, 149.1, 137.1, 132.1, 122.7, 121.6, 119.6, 114.4, 55.2, 48.3. HRMS (ESI):
m/z [M+H]+ calcd for C17H16N3O3, 310.1186; found, 310.1177.
4.2.10.9.
3-((3-Methoxyphenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,2-dione
1
(6i). Off-white solid, yield 52.4% (two steps). mp 197-199 °C. H NMR (400 MHz,
DMSO d6) δ 9.84 (brs, 1H), 8.60 (d, J = 4.2 Hz, 1H), 8.20 (brs, 1H), 7.83 (td, J = 7.7,
1.8 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.35 (dd, J = 7.1, 5.2 Hz, 1H), 7.23 (t, J = 8.1
Hz, 2H), 6.92 (dd, J = 7.9, 1.7 Hz, 1H), 6.60 (dd, J = 8.1, 2.3 Hz, 1H), 4.94 (d, J = 5.5
Hz, 2H), 3.75 (s, 3H). 13C NMR (100 MHz, DMSO d6) δ 184.0, 180.5, 169.0, 163.6,
160.1, 156.8, 149.1, 140.2, 137.1, 130.1, 122.7, 121.7, 110.1, 108.3, 103.7, 55.0, 48.4.
HRMS (ESI): m/z [M+H]+ calcd for C17H16N3O3, 310.1186; found, 310.1183.
4.2.10.10.

3-((2-Methoxyphenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,2-dione
1
(6j). Off-white solid, yield 45.3% (two steps). mp 126-128 °C. H NMR (400 MHz,
DMSO d6) δ 9.37 (brs, 1H), 8.81 (brs, 1H), 8.60 (d, J = 4.5 Hz, 1H), 7.83 (t, J = 7.3
Hz, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.40 – 7.29 (m, 1H), 7.06
– 6.99 (m, 2H), 6.95 – 6.90 (m, 1H), 4.95 (d, J = 5.7 Hz, 2H), 3.88 (s, 3H). 13C NMR
(100 MHz, DMSO d6) δ 184.9, 180.9, 169.7, 164.1, 157.7, 149.8, 149.2, 137.7,
128.2, 124.1, 123.3, 122.3, 121.1, 120.4, 111.7, 56.3, 49.0. HRMS (ESI): m/z [M+H]+
calcd for C17H16N3O3, 310.1186; found, 310.1182.
4.2.10.11.
3-((4-Chlorophenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,2-dione
1
(6k). Off-white solid, yield 54.2% (two steps). mp 240-242 . H NMR (400 MHz,
DMSO d6) δ 9.91 (brs, 1H), 8.60 (d, J = 4.3 Hz, 1H), 8.21 (brs, 1H), 7.83 (td, J = 7.7,
1.7 Hz, 1H), 7.51 – 7.41 (m, 3H), 7.37 (d, J = 8.9 Hz, 2H), 7.36 – 7.32 (m, 1H), 4.93
13
(d, J = 4.8 Hz, 2H). C NMR (100 MHz, DMSO d6) δ 184.6, 181.0, 169.7, 163.9,
157.3, 149.7, 138.5, 137.7, 129.7, 127.0, 123.3, 122.2, 120.1, 49.0. HRMS (ESI): m/z
[M+H]+ calcd for C16H13ClN3O2, 314.0691; found, 314.0690.
4.2.10.12.
3-((Pyridin-2-ylmethyl)amino)-4-((4-(trifluoromethyl)phenyl)amino)cyclobut-3-ene-1,
1
2-dione (6l). Yellow solid, yield 65.1% (two steps). mp 246-248 . H NMR (400
MHz, DMSO d6) δ 10.00 (brs, 1H), 8.60 (d, J = 4.2 Hz, 1H), 8.25 (brs, 1H), 7.84 (td,
J = 7.6, 1.5 Hz, 1H), 7.53 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 7.8 Hz, 1H), 7.38 – 7.33 (m,
3H), 4.94 (d, J = 4.7 Hz, 2H). 13C NMR (100 MHz, DMSO d6) δ 184.2, 180.4, 169.2,
1
163.4, 156.8, 149.1, 143.2, 138.3, 137.1, 122.7, 122.3, 121.7, 120.0 (q, JF, C = 254
Hz), 119.3, 48.4. HRMS (ESI): m/z [M+H]+ calcd for C17H13F3N3O2, 347.0882; found,
347.0890.
4.2.10.13.
3-((6-Morpholinopyridin-3-yl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,
2-dione (6m). Light green solid, yield 71.7% (two steps). mp > 250 °C. 1H NMR (500
MHz, DMSO d6) δ 9.75 (brs, 1H), 8.60 (s, 1H), 8.17 – 8.08 (m, 2H), 7.83 (s, 1H),
7.77 (s, 1H), 7.42 (d, J = 7.1 Hz, 1H), 7.35 (s, 1H), 6.87 (d, J = 8.6 Hz, 1H), 4.92 (s,

2H), 3.69 (brs, 4H), 3.38 (brs, 4H). 13C NMR (100 MHz, DMSO d6) δ 183.4, 180.8,
168.7, 163.7, 157.0, 155.7, 149.1, 137.9, 137.1, 129.0, 127.0, 122.6, 121.6, 107.2,
65.8, 48.3, 45.4. HRMS (ESI): m/z [M+H]+ calcd for C19H20N5O3, 366.1561; found,
366.1553.
4.2.10.14.
3-((5-Morpholinopyridin-2-yl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,
2-dione (6n). Yellow solid, yield 55.6% (two steps). mp > 250 . 1H NMR (400 MHz,
DMSO d6) δ 9.78 (brs, 1H), 8.59 (d, J = 4.1 Hz, 1H), 8.15 (d, J = 2.8 Hz, 2H), 7.83
(td, J = 7.7, 1.8 Hz, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.34 (ddd,
J = 7.5, 4.9, 1.0 Hz, 1H), 6.86 (d, J = 9.1 Hz, 1H), 4.91 (d, J = 4.6 Hz, 2H), 3.71 –
3.67 (m, 4H), 3.41 – 3.35 (m, 4H). 13C NMR (100 MHz, DMSO d6) δ 183.5, 180.9,
168.8, 163.8, 157.1, 155.8, 149.2, 138.0, 137.2, 129.1, 127.2, 122.7, 121.7, 107.3,
65.9, 48.4, 45.6. HRMS (ESI): m/z [M+H]+ calcd for C19H20N5O3, 366.1561; found,
366.1562.
4.2.10.15.
3-((4-(1,4-Oxazepan-4-yl)phenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-en
1
e-1,2-dione (6o). Yellow solid, yield 56.7% (two steps). mp > 250 °C. H NMR (400
MHz, DMSO d6) δ 9.62 (brs, 1H), 8.60 (d, J = 4.3 Hz, 1H), 8.03 (brs, 1H), 7.83 (t, J
= 7.6 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.37 – 7.32 (m, 1H), 7.26 (d, J = 8.0 Hz, 2H),
6.73 (d, J = 7.8 Hz, 2H), 4.98 – 4.83 (m, 2H), 3.74 – 3.63 (m, 2H), 3.59 – 3.42 (m,
13
6H), 1.92 – 1.82 (m, 2H). C NMR (100 MHz, DMSO d6) δ 182.8, 180.7, 168.2,
163.6, 157.1, 149.1, 144.3, 137.1, 128.0, 122.6, 121.6, 119.9, 112.1, 68.7, 68.4, 51.2,
48.3, 47.2, 28.5. HRMS (ESI): m/z [M+H]+ calcd for C21H23N4O3, 379.1765; found,
379.1759.
4.2.10.16.
3-((4-(3-Oxomorpholino)phenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene
-1,2-dione (6p). Yellow solid, yield 40.4% (two steps). mp 250-252 °C. 1H NMR (400
MHz, DMSO d6) δ 9.90 (brs, 1H), 8.61 (d, J = 4.4 Hz, 1H), 8.22 (brs, 1H), 7.84 (t, J
= 7.6 Hz, 1H), 7.45 (t, J = 7.7 Hz, 3H), 7.38 – 7.33 (m, 3H), 4.94 (d, J = 4.5 Hz, 2H),

4.19 (s, 2H), 3.99 – 3.93 (m, 2H), 3.73 – 3.68 (m, 2H). 13C NMR (100 MHz,
DMSO d6) δ 183.9, 180.4, 169.0, 165.8, 163.5, 156.8, 149.1, 137.1, 137.1, 136.3,
126.4, 122.7, 121.7, 118.2, 67.6, 63.4, 48.9, 48.4. HRMS (ESI): m/z [M+H]+ calcd for
C20H19N4O4, 379.1401; found, 379.1402.
4.2.10.17.
3-((4-(1-Oxidothiomorpholino)phenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-
3-ene-1,2-dione (6q). Off-white solid, yield 55.6% (two steps). mp > 250 . 1H NMR
(400 MHz, DMSO d6) δ 9.73 (brs, 1H), 8.60 (d, J = 4.3 Hz, 1H), 8.11 (brs, 1H), 7.83
(td, J = 7.7, 1.7 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.37 – 7.31 (m, 3H), 7.01 (d, J =
9.0 Hz, 2H), 4.92 (d, J = 5.1 Hz, 2H), 3.76 – 3.66 (m, 2H), 3.57 – 3.48 (m, 2H), 2.97
– 2.88 (m, 2H), 2.73 – 2.65 (m, 2H). 13C NMR (100 MHz, DMSO d6) δ 183.5, 180.9,
168.8, 163.9, 157.3, 149.4, 145.4, 137.4, 131.3, 122.9, 121.9, 119.7, 117.0, 48.6, 43.9,
40.6. HRMS (ESI): m/z [M+H]+ calcd for C20H21N4O3S, 397.1329; found, 397.1327.
4.2.10.18.
3-((3-Thiomorpholinophenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,
1
2-dione (6r). White solid, yield 82.5% (two steps). mp 204-206 °C. H NMR (500
MHz, DMSO d6) δ 9.74 (brs, 1H), 8.60 (s, 1H), 8.18 (brs, 1H), 7.83 (s, 1H), 7.43 (d,
J = 7.1 Hz, 1H), 7.35 (s, 1H), 7.16 (dd, J = 17.6, 9.5 Hz, 2H), 6.67 (d, J = 6.0 Hz, 1H),
13
6.59 (d, J = 6.2 Hz, 1H), 4.93 (s, 2H), 3.62 – 3.54 (m, 4H), 2.68 – 2.60 (m, 4H). C
NMR (100 MHz, DMSO d6) δ 183.7, 180.6, 168.9, 163.7, 156.9, 150.9, 149.1, 139.8,
137.1, 129.9, 122.7, 121.6, 110.2, 107.9, 105.3, 50.5, 48.4, 24.8. HRMS (ESI): m/z
[M+H]+ calcd for C20H21N4O2S, 381.1380; found, 381.1380.
4.2.10.19.
3-((3-Methyl-4-morpholinophenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-e
ne-1,2-dione (6s). Off-white solid, yield 81.0% (two steps). mp 240-242 °C. 1H NMR
(500 MHz, DMSO d6) δ 9.73 (brs, 1H), 8.60 (s, 1H), 8.10 (brs, 1H), 7.83 (s, 1H),
7.43 (d, J = 7.2 Hz, 1H), 7.35 (s, 1H), 7.24 (s, 2H), 7.02 (d, J = 8.6 Hz, 1H), 4.93 (s,
2H), 3.72 (s, 4H), 2.79 (s, 4H), 2.25 (s, 3H). 13C NMR (100 MHz, DMSO d6) δ
183.6, 180.6, 168.7, 163.7, 156.9, 149.1, 146.5, 137.1, 134.2, 133.0, 122.7, 121.6,
120.7, 119.7, 116.4, 66.5, 52.0, 48.3, 17.6. HRMS (ESI): m/z [M+H]+ calcd for

C21H23N4O3, 379.1765; found, 379.1765.
4.2.10.20.
3-((3-Methyl-4-thiomorpholinophenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-
1
3-ene-1,2-dione (6t). Off-white solid, yield 84.5% (two steps). mp 235-237 . H
NMR (400 MHz, DMSO d6) δ 9.72 (brs, 1H), 8.66 – 8.52 (m, 1H), 8.10 (brs, 1H),
7.83 (td, J = 7.7, 1.7 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.35 (dd, J = 7.3, 5.0 Hz, 1H),
7.27 – 7.20 (m, 2H), 7.01 (d, J = 9.3 Hz, 1H), 4.92 (d, J = 5.0 Hz, 2H), 3.04 – 2.99 (m,
4H), 2.76 – 2.71 (m, 4H), 2.21 (s, 3H). 13C NMR (100 MHz, DMSO d6) δ 183.6,
180.6, 168.7, 163.7, 156.9, 149.1, 147.8, 137.1, 134.3, 133.4, 122.7, 121.6, 120.6,
120.5, 116.4, 54.1, 48.3, 27.7, 17.5. HRMS (ESI): m/z [M+H]+ calcd for
C21H23N4O2S, 395.1534; found, 395.1536.
4.2.10.21.
3-((3-Cyano-4-morpholinophenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-en
1
e-1,2-dione (6u). Off-white solid, yield 86.3% (two steps). mp > 250 . H NMR
(400 MHz, DMSO d6) δ 9.94 (brs, 1H), 8.60 (d, J = 4.5 Hz, 1H), 8.20 (brs, 1H), 7.86
– 7.79 (m, 2H), 7.55 (d, J = 8.9 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.35 (dd, J = 7.4,
4.9 Hz, 1H), 7.20 (dd, J = 8.8, 2.5 Hz, 1H), 4.93 (d, J = 5.8 Hz, 2H), 3.32 – 3.26 (m,
13
4H), 2.80 – 2.74 (m, 4H). C NMR (100 MHz, DMSO d6) δ 183.9, 180.6, 168.9,
163.1, 156.7, 151.4, 149.1, 137.1, 134.0, 124.0, 122.7, 121.7, 121.3, 117.5, 106.7,
54.2, 48.4, 27.3. HRMS (ESI): m/z [M+H]+ calcd for C21H20N5O2S, 406.1332; found,
406.1330.
4.2.10.22.
3-((Pyridin-2-ylmethyl)amino)-4-((4-thiomorpholino-3-(trifluoromethyl)phenyl)amino
)cyclobut-3-ene-1,2-dione (6v). Off-white solid, yield 72.8% (two steps). mp
1
145-147 . H NMR (400 MHz, DMSO d6) δ 10.03 (brs, 1H), 8.60 (d, J = 4.1 Hz,
1H), 8.20 (brs, 1H), 7.84 (td, J = 7.7, 1.6 Hz, 1H), 7.79 (s, 1H), 7.63 (dd, J = 8.7, 2.4
Hz, 1H), 7.54 (d, J = 8.6 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.35 (dd, J = 7.4, 4.9 Hz,
13
1H), 4.94 (d, J = 4.4 Hz, 2H), 3.06 – 2.99 (m, 4H), 2.73 – 2.66 (m, 4H). C NMR
(100 MHz, DMSO d6) δ 184.1, 180.5, 169.1, 163.2, 156.7, 149.1, 147.2, 137.1,

136.5, 126.8, 126.6, 123.5 (q, 1JF, C = 272 Hz), 122.7, 122.5, 121.7, 116.0, 55.1, 48.4,
27.6, 18.5. HRMS (ESI): m/z [M+H]+ calcd for C21H20F3N4O2S, 449.1254; found,
449.1249.
4.2.10.23.
3-((3-Methoxy-4-thiomorpholinophenyl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobu
1
t-3-ene-1,2-dione (6w). Off-white solid, yield 44.3% (two steps). mp 250-252 °C. H
NMR (500 MHz, DMSO d6) δ 9.78 (brs, 1H), 8.60 (d, J = 4.2 Hz, 1H), 8.13 (brs,
1H), 7.83 (t, J = 7.3 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.37 – 7.33 (m, 1H), 7.28 (s,
1H), 6.88 (d, J = 8.4 Hz, 1H), 6.81 (d, J = 10.1 Hz, 1H), 4.93 (s, 2H), 3.78 (s, 3H),
3.12 (brs, 4H), 2.71 (brs, 4H). 13C NMR (100 MHz, DMSO d6) δ 183.4, 180.7, 168.7,
163.5, 156.9, 152.8, 149.1, 137.4, 137.1, 134.6, 122.7, 121.6, 119.7, 109.7, 103.0,
55.3, 53.0, 48.4, 27.4. HRMS (ESI): m/z [M+H]+ calcd for C21H23N4O3S, 411.1485;
found, 411.1480.
4.2.10.24.
3-((2,3-Dihydro-1H-inden-5-yl)amino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-
1
1,2-dione (6aa). Off-white solid, yield 81.4% (two steps). mp 236-238 . H NMR
(400 MHz, DMSO d6) δ 9.76 (brs, 1H), 8.60 (d, J = 4.8 Hz, 1H), 8.13 (brs, 1H), 7.83
(td, J = 7.7, 1.6 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.35 (dd, J = 7.6, 5.0 Hz, 2H), 7.16
(s, 2H), 4.93 (d, J = 5.5 Hz, 2H), 2.81 (dt, J = 14.9, 7.4 Hz, 4H), 2.00 (p, J = 7.3 Hz,
2H). 13C NMR (100 MHz, DMSO d6) δ 183.7, 180.6, 168.7, 163.8, 157.0, 149.1,
144.9, 138.1, 137.1, 124.6, 122.7, 121.6, 116.2, 114.3, 109.4, 48.4, 32.4, 31.5, 25.1.
HRMS (ESI): m/z [M+H]+ calcd for C19H18N3O2, 320.1393; found, 320.1391.
4.2.10.25.
3-(Benzo[d][1,3]dioxol-5-ylamino)-4-((pyridin-2-ylmethyl)amino)cyclobut-3-ene-1,2-
1
dione (6ab). Brown solid, yield 77.3% (two steps). mp 218-220 . H NMR (400
MHz, DMSO d6) δ 9.77 (brs, 1H), 8.60 (d, J = 4.7 Hz, 1H), 8.12 (brs, 1H), 7.83 (t, J
= 7.6 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 6.4 Hz, 1H), 7.25 (s, 1H), 6.88 (d,
13
J = 8.4 Hz, 1H), 6.75 (d, J = 8.3 Hz, 1H), 6.00 (s, 2H), 4.92 (d, J = 4.0 Hz, 2H). C
NMR (100 MHz, DMSO d6) δ 183.4, 180.6, 168.6, 163.5, 156.9, 149.1, 147.8, 142.9,