Identification of Novel Fused Heteroaromatics-Based MALT1 Inhibitors by High-Throughput Screening to Treat B Cell Lymphoma

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Identiﬁcation of Novel Fused Heteroaromatics-Based MALT1

Inhibitors by High-Throughput Screening to Treat B Cell Lymphoma

Xuewu Liang,^∥Chenxia Sun,^∥Chunpu Li,^∥Haolan Yu,^∥Xiaohui Wei, Xuyi Liu, Wei Bao, Yuqiang Shi,

Xiaochen Sun, Mirzadavlat Khamrakulov, Chenghua Yang,* and Hong Liu *

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ABSTRACT: Development of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) inhibitors is of

great value and signiﬁcance in the treatment of neoplastic disorders and inﬂammatory and autoimmune diseases. However, there is a

lack of eﬀective MALT1 inhibitors in clinic. Herein, a novel class of potent 5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-

a]quinazoline-based MALT1 inhibitors and their covalent derivatives were ﬁrst identiﬁed and designed through high-throughput

screening. We demonstrated that compounds 15c, 15e, and 20c eﬀectively inhibited the MALT1 protease and displayed selective

cytotoxicity to activated B cell-like diﬀuse large B cell lymphoma with low single-digit micromolar potency. Furthermore, compound

20c speciﬁcally repressed NF-κB signaling and induced cell apoptosis in MALT1-dependent TMD8 cells in a dose-dependent

manner. More importantly, 20c showed good pharmacokinetic properties and antitumor eﬃcacy with no signiﬁcant toxicity in the

TMD8 xenograft tumor model. Collectively, this study provides valuable lead compounds of MALT1 inhibitors for further structural

optimization and antitumor mechanism study.

diseases that depend on the activity of MALT1.⁷Thus, the

development of MALT1 inhibitors is of great value and

signiﬁcance in the treatment of cancers and other MALT1-

related diseases.

INTRODUCTION

■

Mucosa-associated lymphoid tissue lymphoma translocation

protein 1 (MALT1) is a key adaptor molecule in the NF-κB

signaling pathway, which is involved in the expression of genes

required for the generation of immune responses, lymphocyte

Full-length human MALT1 possessed a death domain (DD),

the paracaspase domain, and Ig-like domains.⁸Its structure

activation, and lymphocyte development.¹⁻³The early

determined that MALT1 possessed dual biological functions,

literature reported that a fusion oncoprotein MALT1-API2

as a scaﬀolding protein and as a protease (Figure 1). In the

MALT1-mediated NF-κB pathway, TCR and BCR signaling

(cellular inhibitor of apoptosis 2) was discovered in the B cell

neoplasm MALT lymphoma.⁴Blocking MALT1 protease

pathways are the canonical pathways. Upon stimulation, BCR

activity could represent a novel treatment approach for

refractory t (11;18)-positive MALT lymphoma.⁵In addition,

research also showed that MALT1 protease activity plays a key

and TCR signaling indirectly activated PKCβ and PKCθ,

respectively, which led to phosphorylation of the CARMA/

role in the survival and proliferation of activated B cell-like

Received: March 15, 2021

Published: June 28, 2021

diﬀuse large B cell lymphoma (ABC-DLBCL) cells. Inhibition

of MALT1 protease activity can inhibit the growth of MALT1-

dependent ABC-DLBCL by interfering with NF-κB signaling.⁶

Beyond B cell lymphoma, inhibition of MALT1 protease

activity may provide potential eﬀective treatment for other

neoplastic disorders and inﬂammatory and autoimmune

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Figure 1. MALT1-mediated NF-κB pathway and mutation sites of NF-κB signaling in ABC-DLBCL.

Figure 2. Chemical structures of reported small-molecule MALT1 inhibitors.

CARD family and the formation of a CARMA1-BCL10-

MALT1 (CBM) complex. MALT1 assembles into the CBM

complex and functions as a scaﬀolding protein, which

promotes the recruitment of downstream signaling molecules

such as an IKK complex and leads to the activation of the NF-

κB signaling pathway.⁶In addition, MALT1 is a protease that

can cleave its protein substrates such as BCL10, A20, CYLD,

and RelB after a positively charged Arg residue.⁹⁻¹²It should

be noted that the ubiquitin-editing enzyme A20 and

deubiquitinating enzyme CYLD both negatively regulate NF-

κB signaling.^13,14Thus, inhibition of MALT1 protease activity

may lead to the up-regulation of A20 and CYLD and partly

result in repressing NF-κB signaling in the tumor cells.

Activated B cell-like diﬀuse large B cell lymphoma (ABC-

DLBCL) is the most aggressive subtype of diﬀuse large B cell

lymphoma with the highest rate of complications and

mortality.^15,16The key feature of ABC-DLBCL is dependent

on NF-κB signaling mediated by the CBM complex. ABC-

DLBCL tumors abnormally activate NF-κB signaling by

acquiring somatic mutations, such as CD79A/B ITAM

mutation, CARD11/CARMA1 coiled-coil mutation, A20

mutation, and MYD-88 mutation (Figure 1).¹⁷In the aberrant

NF-κB signaling, MALT1 as a component of the CBM

complex helps activate IκB kinase (IKK) and downstream

signaling molecules that lead to NF-κB activation. In addition,

MALT1 potentiates NF-κB signaling by cleaving and

inactivating two negative regulators of IKK, A20 and

CYLD.¹⁷It has been shown that inhibition of MALT1

protease activity was selectively toxic to ABC-DLBCL but not

to germinal center B cell-like (GCB-)DLBCL.⁶Meanwhile, in

the CARD11-BCL10-MALT1 complex, MALT1 is the only

human paracaspase.¹⁸Therefore, the development of MALT1

inhibitors could be a promising approach for activated B cell-

like diﬀuse large B cell lymphoma (ABC-DLBCL) treatment.

So far, a few MALT1 inhibitors were developed and only

one MALT1 inhibitor was developed into the clinical research

stage. The ﬁrst reported speciﬁc MALT1 inhibitor is z-VRPR-

FMK, which is a polypeptide composed of four amino acids.¹⁹

However, this compound is not suitable for clinical study due

to its poor membrane permeability and low bioactivity.⁶Later,

small-molecule MALT1 inhibitors were developed (Figure 2).

The phenothiazine derivative mepazine (1) was originally used

as an antipsychotic drug and was later found to be a

noncovalent selective MALT1 protease inhibitor. Mepazine

possessed a strong inhibition of MALT1 with an IC₅₀of 0.83

μM. Mepazine showed a selective toxicity and induction of

apoptosis in ABC-DLBCL cells in vitro and in vivo.^20,21Despite

such excellent in vitro and in vivo eﬃcacy, side eﬀects such as

tardive dyskinesia limited its clinical research. Melnick et al.

discovered and reported a class of triazole derivatives as

MALT1 inhibitors by biochemical screening in 2012. The

representative compound MI-2 (2) is an irreversible MALT1

inhibitor featuring direct binding to the Cys-464 residue in

MALT1. MI-2 possessed a potent inhibition of MALT1 with

an IC₅₀of 5.84 μM. MI-2 selectively suppressed MALT1-

dependent DLBCL cell lines in vitro and in vivo.²²

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Figure 3. (A) High-throughput screening was initiated to screen a total of 40,000 small molecules. (B) Biochemical activity of 19 compounds

obtained by high-throughput screening. ND indicates “not determined”. LY1 indicates “OCI-LY1”. The antiproliferative activity was shown in a

heat map. The poor and good antiproliferative activities were shown in cyan and orange, respectively. Fold diﬀerence showed the antiproliferative

selectivity to ABC-DLBCL (HBL1 and TMD8). Fold diﬀerence = antiproliferative activity data (OCI-LY1)/mean value of antiproliferative activity

data (HBL1 + TMD8). (C) Scaﬀold structure of the 15 compounds indicated by red compound ID in panel (B).

Furthermore, recent research found that MI-2 was eﬀective in

ibrutinib-resistant chronic lymphocytic leukemia (CLL) and

inﬂammation diseases.²³With the excellent in vitro and in vivo

eﬃcacy, MI-2 as a lead compound attracted more researchers

to carry out structure modiﬁcation.²⁴Hong et al. discovered a

new class of potent covalent β-lapachone-based MALT1

inhibitors (3), which possessed a strong inhibition of

MALT1 with an IC₅₀of 1.6 μM. β-Lapachone-based

MALT1 inhibitors exhibited potent antiproliferative activity

to ABC-DLBCL cells and inhibited the cleavage of CYLD-

mediated MALT1 in vitro.²⁵In 2019, Novartis reported a class

of pyrazolo pyrimidine derivatives (4) as noncovalent MALT1

inhibitors.²⁶Although these derivatives showed a potent

MALT1 inhibition with IC₅₀at the nanomolar level, they

have a preference to binding to and stabilizing mutant MALT1

and restoring MALT1 activity rather than inhibiting the

enzymatic activity of mutant MALT1.²⁷Later, Novartis

reported two representative compounds, that is, MLT-231

and MLT-985, which potently and selectively inhibited the

proteolytic activity of MALT1 in 2020. However, the

structures of these two compounds are very similar to that of

4. Among these two compounds, MLT-985 eﬀectively led to

tumor regression in an ABC-DLBCL lymphoma xenograft

model.^28,29In 2018, Lorena et al. reported a z-VRPR-FMK

derivative 5 as a substrate-mimetic peptidic covalent MALT1

inhibitor. This compound showed maximal inhibition of

MALT1 activity in in vitro and cell-based assays. Additionally,

this compound eﬀectively suppressed the growth of ABC-

DLBCL tumors (TMD8 and OCI-Ly3 xenografts) in mice at a

dose of 30 mg/kg b.i.d.³⁰Despite the excellent in vitro and in

vivo eﬃcacy, the potential poor oral bioavailability and toxicity

caused by its covalent bind to MALT1 protein are not

conducive to the further development of compound 5. It is

worth mentioning that Johnson & Johnson has developed a

case of MALT1 inhibitor JNJ-67856633 into the clinical phase

I study, but the structure of this molecule has not been

revealed until now.

MALT1 inhibitors could be a promising and powerful

approach for activated B cell-like diﬀuse large B cell lymphoma

treatment. However, there have been few research and

development cases of MALT1 inhibitors so far.³¹Therefore,

the development of MALT1 inhibitors is urgent for the

treatment of lymphoma. Herein, we identiﬁed and designed a

novel class of potent 5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo-

[3,4-a]quinazoline-based MALT1 inhibitors and their covalent

derivatives and demonstrated their excellent antitumor eﬀects

on ABC-DLBCL cells.

RESULTS AND DISCUSSION

■

Discovery of Novel MALT1 Inhibitors by High-

Throughput Screening. To develop a new structural class

of potent MALT1 inhibitors, high-throughput screening was

initiated to identify novel small molecules that can potently

inhibit the paracaspase activity of MALT1. In the high-

throughput screening, a ﬂuorogenic assay was developed to

evaluate the MALT1 inhibitory activity of small molecules

using MALT1 substrate peptide LVSR linked to the ﬂuorogen

AMC. Recombinant MALT1 (amino acids 340−789) fused

with a leucine zipper dimerization motif (LZ-MALT1) was

used in the screen as before,²²which promotes its dimerization

and activation. With this assay, a total of 40,000 small

molecules were screened for their inhibitory activity against

MALT1 paracaspase. Using 60% inhibition as a threshold,

1000 candidate compounds were selected for validation in a

concentration−response assay (Figure 3A). Of these, 19

compounds were selected for further validation based on

their biochemical activity. The results in Figure 3B showed that

these 19 compounds possessed potent inhibitory activity

against MALT1 paracaspase with IC₅₀values lower than 22

μM. Importantly, most of these tested compounds showed

potent antiproliferative activity against HBL1 and TMD8

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Figure 4. (A) Three hundred analogues purchased from ChemDiv were screened by LZ-MALT1 ﬂuorogenic assay. (B) Biochemical activity of 17

compounds obtained by the screening of the 300 compounds. ND indicates “not determined”; NA indicates “not active”. LY1 indicates “OCI-LY1”.

The antiproliferative activity was shown in a heat map. The poor and good antiproliferative activities were shown in cyan and orange, respectively.

Fold diﬀerence = antiproliferative activity data (OCI-LY1)/mean value of antiproliferative activity data (HBL1 + TMD8). (C) Structure of

compound F28, which was used in the docking analysis. Phenyl A and B groups are shown in red color. Chemical structures of compounds F1−

F36 are listed in Figure S1, Supporting Information.

ABC-DLBCL cells. By further structural analysis, we found

that 15 compounds (indicated by red compound ID in Figure

3B) had a common scaﬀold, 5-oxo-1-thioxo-4,5-dihydro-1H-

thiazolo[3,4-a]quinazoline (Figure 3C; see the chemical

structures in the Supporting Information).

formed a covalent bond with Cys464 in MALT1 via alkylation.

Reversible noncovalent MALT1 inhibitors, such as the

phenothiazine derivative thioridazine and pyrazolo pyrimidine

derivative 4, bound reversibly at the allosteric site to inactive

MALT1. Through the docking analysis and chemical analysis

(see Figure S2, Supporting Information), we hypothesized that

5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-

based MALT1 inhibitors might be a class of reversible

noncovalent MALT1 inhibitors and acted on the allosteric

site in MALT1. Therefore, we rationally designed the novel

MALT1 inhibitors based on the binding mode between small-

molecule inhibitors and the allosteric site in MALT1.

The phenothiazine derivative thioridazine was the ﬁrst small

molecule found to act on the allosteric site in MALT1. A

detailed binding analysis (PDB code: 4I1R) showed that the

tricyclic ring of thioridazine was bound in a hydrophobic

pocket composed of residues Ala394, Phe398, and Leu401 in α

helix and Leu346, Val344, and Val381 in β-sheets 1 and 2,

respectively (Figure 5A,B). In addition, N-methylpiperidine

nitrogen of thioridazine formed a key hydrogen bond with the

Glu397 residue in MALT1.²¹Through the complex structural

analysis, we selected a representative compound F28 to

progress to the molecular docking analysis (Figure 5C,D). We

found that compound F28 possessed a similar binding mode as

thioridazine. The scaﬀold 5-oxo-1-thioxo-4,5-dihydro-1H-

thiazolo[3,4-a]quinazoline was well ﬁt in the hydrophobic

pocket composed of amino acid residues in α helix and in β-

sheets 1 and 2. Importantly, amide nitrogen near the phenyl A

group formed a key hydrogen bond with the Glu397 residue in

MALT1.

To further establish whether the scaﬀold 5-oxo-1-thioxo-4,5-

dihydro-1H-thiazolo[3,4-a]quinazoline represented a potential

core structure for the development of novel MALT1 inhibitors,

a total of 300 analogues containing the scaﬀold (purchased

from ChemDiv) were screened. The results in Figure 4A

showed that 29 compounds displayed higher than 60%

inhibition against MALT1. Among these compounds, 17

compounds showed potent inhibitory activity against MALT1

with IC₅₀values lower than 10 μM and were selected for

further characterization in cell-proliferation assays. All these

analogues exhibited the same selectivity in the suppression of

the proliferation of the ABC-DLBCL cell lines, with GI₅₀

concentrations in the micromolar range (Figure 4B).

Herein, by a high-throughput screening, we identiﬁed a

novel series of derivatives containing the 5-oxo-1-thioxo-4,5-

dihydro-1H-thiazolo[3,4-a]quinazoline scaﬀold as potent

MALT1 inhibitors. To ﬁnd more potent MALT1 inhibitors

and comprehensively elucidate the structure−activity relation-

ship of them, we next adopted the rational drug design strategy

to design two novel classes of target compounds.

Rational Design of Novel MALT1 Inhibitors. Based on

the diﬀerent modes of action, MALT1 inhibitors were

classiﬁed into two categories, irreversible covalent MALT1

inhibitors and reversible noncovalent MALT1 inhibitors.

Irreversible covalent MALT1 inhibitors, such as z-VRPR-

FMK, MI-2 (2), β-lapachone (3), and z-VRPR-FMK derivative

5, occupied the active pocket in the paracaspase domain and

Through the docking analysis, it can be seen that there is still

some extra space in this hydrophobic pocket that is not yet

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Figure 5. (A, B) Binding mode of thioridazine in the allosteric site in MALT1 (PDB code: 4I1R). (C, D) Calculated binding mode of compound

F28 in the allosteric site in MALT1 (PDB code: 4I1R). In the structure of the ligand, carbon, chlorine, oxygen, nitrogen, sulfur, and polar hydrogen

atoms are shown in green, dark green, red, blue, gold, and white, respectively. Yellow dashed lines indicate hydrogen bonds. The ﬁgures were

generated using PyMol (http://www.pymol.org/).

Figure 6. Rational design of novel MALT1 inhibitors based on the novel scaﬀold identiﬁed by HTS.

completely ﬁlled by the small molecule (Figure 5C). To ﬁnd

more potent MALT1 inhibitors, we carried out rational

structural modiﬁcation based on the scaﬀold structure. First,

we investigated the MALT1 inhibitory activity of the structure

skeleton without any substituents to design compound 14a in

order to prove the importance of the substituents on the two

phenyl groups (Figure 6). Then, to ﬁll the unoccupied pocket

A to build more interactions, diﬀerent substituents were

introduced to the phenyl A group to design compounds 14b−

14k. In addition, we introduced diﬀerent substituents

(electron-withdrawing, electron-donating, single, or multiple,

etc.) to the phenyl B group to ﬁll the unoccupied pocket B and

build more interactions with the amino acid residues. In this

way, we designed compounds 15a−15r. Last, we designed

compounds 16a−16g to ﬁnd more potent MALT1 inhibitors

based on the SAR of the compounds 14a−14k and 15a−15r.

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a

Scheme 1. Synthesis of Target Compounds 14a−14k, 15a−15r, and 16a−16g

a

Reagents and conditions: (a) PCl₅, CH₂Cl₂, 40 °C, overnight, 50−80% yields; (b) NaHCO₃(aq)/H₂O, CH₂Cl₂, r.t., overnight, 58−78% yields;

(c) Et₃N, DMF, 50 °C, overnight, 16−60% yields.

a

Scheme 2. Synthesis of Target Compounds 20a−20f

a

Reagents and conditions: (a) PCl₅, CH₂Cl₂, 40 °C, overnight, 80% yield; (b) Et₃N, EtOH, 50 °C, overnight, 40−60% yields; (c) chloroacetyl

chloride, CH₃CN, 65 °C, microwave, 1 h, 15−33% yields.

Based on the above rational design idea, a total of 36

compounds have been designed and synthesized for this series

of MALT1 inhibitors.

The synthesis of target compounds 20a−20f is described in

Scheme 2. Cyanoacetic acid (6) reacts with 2,5-dimethoxyani-

line to give the intermediate 8i. The condensation of 8i,

isothiocyanatobenzene derivatives 18a−18f, and sulfur (12) by

Gewald’s reaction gives the intermediate compounds 19a−19f.

Intermediate compounds 19a−19f react with chloroacetyl

chloride under the microwave condition to give the target

compounds 20a−20f. The structures of all target compounds

In addition, covalent modiﬁcation strategies are widely used

in the discovery of novel MALT1 inhibitors, such as MI-2 (2),

and β-lapachone-based MALT1 inhibitor (3).^22,25,32Mean-

while, destruction of rigidity in chemical structures is beneﬁcial

to improve the drug-like properties.³³Inspired by this, we

designed another series of covalent MALT1 inhibitors 20a−

20f based on the 5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-

a]quinazoline scaﬀold (Figure 6).

Chemistry. The synthesis of target compounds 14a−14k,

15a−15r, and 16a−16g is described in Scheme 1. Cyanoacetic

acid (6) reacts with various substituted anilines (7) to obtain

the cyanamide derivatives 8 by amide condensation. Various

substituted methyl anthranilate (9) reacts with thiophosgene

(10) under the basic condition to give methyl-2-isothiocyana-

tobenzoates 11. The condensation of cyanamide derivatives 8,

methyl-2-isothiocyanatobenzoates 11, and sulfur (12) by

Gewald’s reaction gives the target compounds 14a−14k,

15a−15r, and 16a−16g. It is worth noting that the last step

may proceed to a subsequent intramolecular amide con-

densation reaction of Gewald’s products 2(3H)-thioxo-1,3-

thiazoles 13.³⁴

1

were characterized by HRMS, H NMR, and ¹³C NMR data.

In Vitro MALT1 Inhibition Assay. We then evaluated the

in vitro MALT1 inhibition eﬀect for all synthesized derivatives.

Thioridazine, a reported noncovalent selective MALT1

inhibitor, was used as the positive control.^20,21,31As

summarized in Table 1, we ﬁrst investigated the inhibition

eﬀect of the structure skeleton with diﬀerent substituents on

MALT1 protease activity. Compound 14a, the structure

skeleton with a hydrogen atom substituent, showed a weak

in vitro MALT1 inhibition with the IC₅₀value over 25 μM.

This result further conﬁrmed the importance of the

substituents on the two phenyl groups. To deeply investigate

the eﬀect of the R¹substituent on the inhibitory activity of

MALT1, we introduced diversiﬁed R¹groups and continued to

design and synthesize compounds 14b−14k. The results in

Table 1 showed that the compounds 14a−14e, which

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Table 1. Potency of 5-Oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline Derivatives at Inhibiting MALT1 Protease

a

Activity

a

Data are mean values of three independent experiments performed in replicate (n ≥ 3); IC₅₀values are shown as mean SD.

contained the electron-donating groups and electron-with-

drawing groups on the phenyl group in the R¹substituent,

showed the poor inhibitory activity against MALT1, except

compound 14b, which contained para-nitro on the phenyl

group in the R¹substituent. It was noted that compound 14b

showed the potent inhibition on MALT1 protease activity with

an IC₅₀of 6.7 μM. In addition, compounds 14e−14h, which

contained para-, meta-, and ortho-methoxy groups and

polymethoxy groups on the phenyl group in the R¹substituent,

all showed poor inhibitory activity on MALT1 (IC₅₀> 25

μM). The in vitro MALT1 inhibitory activity of compounds

14j and 14k, which contained dichloro groups at diﬀerent sites

on the phenyl group in the R¹substituent, varied greatly.

Compound 14j, which contained 3,5-dichloro on the phenyl

group, showed potent in vitro MALT1 inhibitory activity with

an IC₅₀of 2.2 μM, while compound 14k, which contained 3,4-

dichloro on the phenyl group, possessed weak bioactivity. It

was noted that compound 14i, which contained ortho-ethyl on

the phenyl group in the R¹substituent, showed a moderate

inhibition on MALT1 protease activity with an IC₅₀of 14.7

μM. From the results in Table 1, it is easy to ﬁgure out that

minor modiﬁcation of the R¹substituent leads to a signiﬁcant

change in MALT1 inhibitory activity.

The type of R¹substituent was shown to be very important

to the bioactivity of these analogues in Table 1. In the high-

throughput screening, we found that the compounds

containing 2,5-dimethoxyphenyl in the R¹group all showed

potent inhibitory activity against MALT1 paracaspase, such as

compounds F3, F26, F28, and F31 (Figure 3B; see the

chemical structures in the Supporting Information). Based on

the above result of the high-throughput screening, we kept R¹

unchanged (2,5-dimethoxyphenyl in the R¹group) and

continued to design compounds 15a−15j to further investigate

the eﬀect of R²and R³substituents on the inhibitory activity of

MALT1. The results in Table 2 showed that R²and/or R³with

electron-donating groups would be detrimental to the activity

of the compounds (compounds 15a, 15f, and 15j). On the

contrary, R²and/or R³with electron-withdrawing groups

would be beneﬁcial for compounds to bind to the MALT1

protease (compounds 15b−15e and 15g−15i). Through this

round of structural modiﬁcation and in vitro MALT1 inhibition

assay, we obtained many potent MALT1 inhibitors with IC₅₀

values in the single-digit μM range. Especially, compounds 15c,

15e, and 15h potently inhibited MALT1 with the IC₅₀values

of 5.2, 5.3, and 3.2 μM, respectively. It was noted that the

structural modiﬁcation of R¹, R², and R³groups of 14a led to

compounds 15c, 15e, and 15h with the potent activity against

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Table 2. Potency of 5-Oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline Derivatives at Inhibiting MALT1 Protease

a

Activity

a

Data are mean values of three independent experiments performed in replicate (n ≥ 3); IC₅₀values are shown as mean SD.

MALT1. From the results in Table 1, para-nitro and 3,5-

dichloro on the phenyl group in the R¹substituent would be

beneﬁcial for MALT1 inhibitory activity. Therefore, we

continued to design compounds 15k−15r, which contained

para-nitro, 3,5-dichloro, and 3-chloro-2-nitro on the phenyl

group in the R¹substituent. As expected, compounds 15k−15r

showed potent MALT1 inhibitory activity with IC₅₀values of

2.5−7.4 μM, except that compound 15p, which contained the

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a

Table 3. Potency of Novel Covalent Derivatives at Inhibiting MALT1 Protease Activity

compd.

20a

20b

20c

thioridazine

R²

R³

MALT1, IC₅₀(μM)

compd.

R²

R³

MALT1, IC₅₀(μM)

CN

CF₃

H

CF₃

5.5 1.9

3.2 1.4

5.3 1.3

4.1 1.5

20d

20e

20f

H

F

Br

Cl

H

7.0 0.8

3.8 1.3

4.6 0.9

a

Data are mean values of three independent experiments performed in replicate (n ≥ 3); IC₅₀values are shown as mean SD.

Table 4. Antiproliferative Activity of 5-Oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline and Its Covalent Derivatives

a

against B Cell Lymphoma

GI₅₀(μM)

b

compd.

14b

14j

15c

15e

MALT1, IC₅₀(μM)

HBL1

TMD8^b

OCI-LY1^b

fold diﬀerence^c

6.7 2.8

2.2 0.6

5.2 2.3

5.3 1.3

6.9 3.5

3.2 0.5

3.3 1.9

5.1 1.7

5.5 1.9

3.2 1.4

5.3 1.3

7.0 0.8

3.8 1.3

4.6 0.9

4.1 1.5

14.3 5.7

8.0 1.0

8.2 0.6

6.7 1.8

11.7 0.9

14.0 1.9

18.4 3.4

14.8 1.4

1.7 0.1

2.2 0.6

1.0 0.2

1.5 0.2

1.6 0.1

1.8 0.3

6.6 0.3

4.0 1.1

3.7 1.0

8.4 1.3

7.0 0.8

11.0 1.8

12.4 0.7

3.9 1.4

4.4 1.5

1.6 0.3

1.3 0.4

0.4 0.1

1.0 0.3

1.5 0.1

1.2 0.1

5.9 1.3

21.0 5.5

10.2 2.2

20.7 2.5

13.4 2.4

12.9 5.1

28.1 1.7

20.2 5.1

18.6 2.8

11.8 0.3

13.3 0.3

10.7 0.2

13.0 0.2

12.1 0.2

12.7 0.1

13.4 2.1

1.5

1.7

2.5

2.0

1.1

2.1

1.7

1.9

7.1

7.6

15.2

10.4

7.8

8.4

15g

15h

15n

16e

20a

20b

20c

20d

20e

20f

thioridazine

2.1

a

b

Data are mean values of three independent experiments performed in replicate (n ≥ 3); IC₅₀and GI₅₀values are shown as mean SD. HBL1

c

and TMD8 cancer cells belong to ABC-DLBCL. OCI-LY1 cancer cells belong to GCB-DLBCL. Fold diﬀerence = antiproliferative activity data

(OCI-LY1)/mean value of antiproliferative activity data (HBL1 + TMD8).

methoxy group in R²and R³, showed mild MALT1 inhibitory

activity with an IC₅₀of 14.0 μM, further proving that R²and R³

with electron-donating groups would be detrimental to the

activity of the compounds.

that structural modiﬁcation on the R¹group would lead to

more potent MALT1 inhibitors with structural diversity.

Next, we continued to evaluate the potency of novel

covalent derivatives 20a−20f at inhibiting MALT1 protease

activity. The results in Table 3 showed that these novel

covalent derivatives possessed the potent MALT1 inhibitory

activity with the IC₅₀values in the single-digit μM range.

Among these compounds, 20b, 20c, and 20e exhibited potent

MALT1 inhibitory activity with the IC₅₀values of 3.2, 5.3, and

3.8 μM, respectively, which further conﬁrmed the rationality of

the covalent modiﬁcation strategy in the design of MALT1

inhibitors.

Eﬀects on the Proliferation of B Cell Lymphoma. The

target compounds with excellent MALT1 inhibitory activities

were further tested for their growth inhibition eﬀect on B cell

lymphoma cells, including ABC-DLBCL cell lines HBL1 and

TMD8 and the GCB-DLBCL cell line OCI-LY1. The results

are shown in Table 4. We observed that 5-oxo-1-thioxo-4,5-

dihydro-1H-thiazolo[3,4-a]quinazoline derivatives clearly re-

pressed cell growth in HBL1 and TMD8 cells in a dose-

dependent manner. These compounds displayed potent

cytotoxicity to ABC-DLBCL cells at the micromolar level.

Especially, compounds 15c and 15e showed selective toxicity

to ABC-DLBCL (HBL1 and TMD8) rather than GCB-

To obtain more potent MALT1 inhibitors, we designed and

synthesized more 5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-

a]quinazoline derivatives based on the above SARs and the

results of high-throughput screening. The inhibitory activity

results of compounds 16a−16g showed that the compounds

with various R¹, R², and R³groups possessed the potent

MALT1 inhibitory activity with the IC₅₀values in the single-

digit μM range. The MALT1 inhibitory activities of 5-oxo-1-

thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline derivatives

are equal to that of the positive control thioridazine. In

summary, we ﬁrst identiﬁed a novel scaﬀold, 5-oxo-1-thioxo-

4,5-dihydro-1H-thiazolo[3,4-a]quinazoline as a potent MALT1

inhibitor. From the results of Tables 1 and 2, it can be seen

that the minor modiﬁcation of the R¹, R², and R³groups would

largely aﬀect the MALT1 inhibitory activity of 5-oxo-1-thioxo-

4,5-dihydro-1H-thiazolo[3,4-a]quinazoline derivatives. Gener-

ally, R²and/or R³with electron-withdrawing groups (such as

Cl, Br, F, and ester group) would be beneﬁcial for the MALT1

inhibitory activity of these analogues. Diﬀerent substituents in

the R¹group were well tolerated, which further demonstrated

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Figure 7. (A) Induction of apoptosis in HBL-1, TMD8, and OCI-LY1 cancer cells after 24 h treatment with compound 20c. (B) Quantitation of

the apoptotic cells induced by compound 20c after 24 h treatment in HBL1, TMD8, and OCI-LY1 cells. Data are expressed as the mean

standard error (n = 3). “ns” indicates not signiﬁcant; **P < 0.01, and ***P < 0.001 vs control.

DLBCL (OCI-LY1) to some extent (about 2-fold selectivity).

Meanwhile, the novel covalent derivatives 20a−20f displayed

potent cytotoxicity to ABC-DLBCL cells at the low micro-

molar level. Among these compounds, 20c, as the

representative, possessed the most potent antiproliferative

activity against ABC-DLBCL HBL1 and TMD8 cells with the

GI₅₀values of 1.0 and 0.4 μM, respectively. Note that

compound 20c showed selective toxicity to ABC-DLBCL

(HBL1 and TMD8) rather than GCB-DLBCL (OCI-LY1)

with 15.2-fold selectivity, which further conﬁrmed that

MALT1 inhibitors were more eﬀective for ABC-DLBCL

than GCB-DLBCL. To our best knowledge, the MALT1

inhibitor 20c probably possessed the most antiproliferative

selectivity (about 15-fold) against ABC-DLBCL rather than

GCB-DLBCL. In addition, the antiproliferative activities of

novel covalent derivatives 20a−20f in ABC-DLBCL cells are

generally superior to that of 5-oxo-1-thioxo-4,5-dihydro-1H-

thiazolo[3,4-a]quinazoline derivatives and the positive control

thioridazine.

Apoptosis Assay. To explore the antiproliferative

mechanisms of these MALT1 inhibitors, we ﬁrst evaluated

whether these 5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]-

quinazoline derivatives induced apoptosis of B cell lymphoma

cells. TMD8 and OCI-LY1 cells were treated with increasing

concentrations of compounds 15c and 15e, respectively. After

24 h, cells were harvested and stained with Annexin V-FITC

and propidium iodide (PI). The apoptosis levels were assessed

by ﬂow cytometry. It was demonstrated that both compounds

15c and 15e could induce apoptosis in TMD8 cells in a dose-

dependent manner but did not induce signiﬁcant apoptosis in

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OCI-LY1 cells (Figure S3). We further evaluated the apoptosis

induced by the covalent MALT1 inhibitor 20c. Results in

Figure 7 demonstrated that 20c could induce apoptosis in

HBL1 and TMD8 cells in a dose-dependent manner, but it had

little eﬀect on OCI-LY1 cell apoptosis, which further

conﬁrmed the selective growth inhibition against ABC-

DLBCL. Remarkably, even at 5 μM, compound 20c showed

over 50% apoptosis in HBL1 and TMD8 cancer cells, which

possessed much more potent pro-apoptotic activity than that

of compounds 15c and 15e (less than 20% apoptosis in HBL1

and TMD8 cancer cells at 5 μM).

In addition, we examined the protein levels of PARP and

caspase-3 in HBL1, TMD8, and OCI-LY1 cells after 12 h

treatment with compound 20c. We found that compound 20c

could induce the cleavage of PARP and caspase 3 in HBL1 and

TMD8 cells in a dose-dependent manner, but it had very little

eﬀect on OCI-LY1 cells (Figure 8), which is consistent with

the pro-apoptotic activity of compound 20c as shown in Figure

7.

Figure 9. Eﬀect of compound 20c on MALT1 protein and NF-κB

signaling in HBL1, TMD8, and OCI-LY1 cancer cells for 12 h. β-

Actin was used as a loading control in the western blot.

kg po routes (Table 5). Compound 20c proved to be suitable

for both oral administration and intraperitoneal administration,

with a high area under the curve (AUC), long half-life (t_1/2),

and good bioavailability (F) in CD-1 mice. To be speciﬁc,

compound 20c displayed a long half-life of 2.83 h, AUC_0−tof

11,162 ng h/mL, and a high oral bioavailability of 110% at 10

mg/kg oral administration in CD-1 mice. The results

conﬁrmed that compound 20c possessed a relatively desired

pharmacokinetic property.

In Vivo Antitumor Activity Evaluation and Determi-

nation of Drug Accumulation. Classic TMD8 cell line-

derived xenografts were used for in vivo eﬃcacy evaluation.

NCG mice bearing TMD8 tumors were randomized and

treated with compound 20c by intraperitoneal administration

at doses of 30 mg/kg for 13 consecutive days. As shown in

Figure 10, compound 20c could suppress tumor growth at 30

mg/kg/day with a TGI value of 31%. In addition, adverse

reactions such as diarrhea, emaciation, and body weight loss

were not observed in mice, which proved that compound 20c

had a relatively good safety proﬁle. Through further intra-

tumoral drug concentration testing, it was found that at a dose

of 30 mg/kg/day, the accumulation of compound 20c for 13

days in the tumor reached 1132 ng/g, which further proved

that compound 20c could reach the tumor tissue smoothly and

accumulate in the tumor.

Docking Analysis of Compound 20c in the Para-

caspase Domain of MALT1. Compound 20c, as a novel and

representative MALT1 inhibitor, was further progressed into a

docking analysis to gain deeper insights into the molecular

basis of the inhibitory activity. The calculated binding mode of

compound 20c in the paracaspase domain of MALT1 is shown

in Figure 11. The results indicated that the key amino residue

Cys464 in the paracaspase domain of MALT1 attacked the

chloroacetamide group in the structure of compound 20c and

formed a covalent bond between compound 20c and MALT1

protease. The key covalent binding leads to an irreversible

blockage of MALT1’s proteolytic activity by compound 20c

with single-digit micromolar potency. In addition, compound

20c was well ﬁtted in the pocket of the MALT1 paracaspase

domain.

Figure 8. Eﬀect of compound 20c on apoptosis in diﬀerent cell lines.

HBL1, TMD8, and OCI-LY1 cells were treated with DMSO or

compound 20c for 12 h. Apoptosis was evidenced by the increase in

cleaved PARP protein levels and decreased caspase-3 protein levels.

GAPDH was used as a loading control.

Inhibitory Eﬀect on MALT1 Activity and NF-κB

Signaling. Western blot analysis was performed to evaluate

the eﬀect of the covalent MALT1 inhibitor 20c on MALT1’s

enzymatic activity and NF-κB signaling at the cellular level.

MALT1 is a protease that can cleave its protein substrates such

as BCL10, A20, CYLD, and RelB when activated. Here, the

level of the cleaved RelB was measured to evaluate the activity

of MALT1. IκBα is the inhibitor of NF-κB, which can bind and

sequester NF-κB in the cytoplasm. When phosphorylated by

the upstream signal, IκBα gets degraded and releases NF-κB,

which leads to NF-κB nucleus translocation and activation.

The level of phosphorylated IκBα could reﬂect the activation

of NF-κB signaling. HBL1, TMD8, and OCI-LY1 cancer cells

were pretreated with increasing concentrations of 20c (4 and 8

μM) for 30 min, followed by the 10 μM proteasome inhibitor

MG-132 for 2 h. Results in Figure 9 showed that at 8 μM,

compound 20c could eﬀectively suppress the cleavage of the

MALT1 substrate RelB in HBL1 and TMD8 cells, which

proved that compound 20c directly inhibited MALT1

paracaspase activity. In addition, compound 20c also repressed

the NF-κB signaling as evidenced by the reduced phosphor-

ylation of IκBα (p-IκBα).

CONCLUSIONS

■

MALT1 inhibitors provide an eﬀective treatment for neoplastic

disorders and inﬂammatory and autoimmune diseases. More

importantly, MALT1 inhibitors could be a promising and

powerful approach for aggressive ABC-DLBCL treatment. In

the present work, we discovered a novel class of potent 5-oxo-

1-thioxo-4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-based

In Vivo Pharmacokinetic (PK) Proﬁle. Due to its potent

inhibitory activity against MALT1 and anticancer activities in

vitro, compound 20c was progressed into the in vivo

pharmacokinetic study. Pharmacokinetic parameters were

determined in mice at 1 mg/kg iv, 1 mg/kg ip, and 10 mg/

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Table 5. PK Parameters of Compound 20c in ICR (CD-1) Mice (n = 9, Male)

dose (mg/kg)

administration

t_1/2(h)

T_max(h)

C_max(ng/mL)

AUC_0−t(ng h/mL)

MRT_0−t(h)

V_z(L/kg)

CL (mL/min/kg)

14.1

F (%)

10

1

po

ip

iv

2.83

4.50

4.60

3.00

0.35

1526

336

11,162

732

1011

5.62

5.95

5.66

110

72

4.82

Figure 10. (A) Growth curve of implanted TMD8 xenograft in nude mice. (B) Body weight curve of the nude mice.

further puriﬁcation. All reactions were monitored by thin-layer

chromatography on 0.25 mm silica gel plates (60 GF-254) and

1

visualized with UV light or iodine vapor. H NMR and ¹³C NMR

were generated in DMSO-d₆, or pyridine-d₅, on Varian Mercury 400,

500, or 600 M NMR spectrometers. Chemical shifts were reported in

parts per million (ppm). Multiplicity of ¹H NMR signals was reported

as single (s), double (d), triplet (t), quarter (q), and multiplet (m).

ESI-MS was determined on an API 4000 spectrometer. The high-

resolution mass spectra were obtained by Shanghai Institute of

Materia Medica. Melting points were determined on an electro-

thermal melting point apparatus and were uncorrected. HPLC

analysis was carried out on an Agilent 1200 with an extended C18

column (4.6 mm × 250 mm, 5 μm). The ﬂow rate was 1 mL/min.

Part ﬁnal compounds were pretreated with an amount of triethyl-

amine as a cosolvent and achieved a minimum of 95% purity.

General Procedure for the Preparation of 8a−8l. 2-Cyano-N-(4-

nitrophenyl)acetamide (8a). To a solution of cyanoacetic acid (6,

1.23 g, 14.5 mmol, 1.0 equiv) in CH₂Cl₂(100 mL) was slowly added

phosphorus pentachloride (3.32 g, 15.9 mmol, 1.1 equiv). The

mixture was stirred at 40 °C for 30 min. Then, 4-nitroaniline (7a, 2.0

g, 14.48 mmol, 1.0 equiv) was slowly added to the reaction solution.

The resulting mixture was heated at 40 °C overnight and a precipitate

appeared. Upon completion of the reaction, the reaction solution was

ﬁltered and the precipitate was collected. The precipitate was

dissolved in ethyl acetate (300 mL). The organic layer was washed

with water (3 × 50 mL) and brine (3 × 50 mL), dried over sodium

sulfate, and concentrated to give compound 8a (1.9 g, 64% yield) as a

Figure 11. (A) Calculated binding mode of compound 20c in the

paracaspase domain of MALT1 (PDB code: 4UO8). (B) Surface

representation of compound 20c in the paracaspase domain of

MALT1 (PDB code: 4UO8). In the ligand, oxygen, nitrogen, carbon,

sulfur, ﬂuorine, and polar hydrogen atoms are shown in red, blue,

brown, gold yellow, pale cyan, and white, respectively. The key amino

residue Cys464 was indicated by the green sticks and formed a

covalent bond with 20c. The ﬁgures were generated using PyMol

(http://www.pymol.org/).

MALT1 inhibitors by high-throughput screening. After several

rounds of structural modiﬁcation based on the scaﬀold

structure, we obtained many potent MALT1 inhibitors with

an IC₅₀value at the micromolar level. Among these MALT1

inhibitors, representative compounds 15c, 15e, and 20c

exhibited potent MALT1 inhibition with IC₅₀values of 5.2,

5.3, and 5.3 μM, respectively, and selectively suppressed the

proliferation of ABC-DLBCL cancer lines HBL1 and TMD8.

In addition, compound 20c eﬀectively induced apoptosis of

TMD8 cells in a dose-dependent manner, inhibited the

cleavage of RelB by MALT1, and repressed the NF-κB signal

in ABC-DLBCL cells. Furthermore, 20c displayed an excellent

pharmacokinetic property and repressed the growth of TMD8

cells in the xenograft tumor model with no signiﬁcant toxicity.

In summary, the merit of our research is the development of a

novel series of MALT inhibitors with excellent in vitro

antitumor activity, which provides valuable lead compounds

for further structural optimization and antitumor mechanism

study.

1

light yellow solid. H NMR (400 MHz, DMSO-d₆) δ 10.94 (s, 1H),

8.27−8.18 (m, 2H), 7.83−7.74 (m, 2H), 4.00 (s, 2H).

N-(4-Chlorophenyl)-2-cyanoacetamide (8b). 4-Chloroaniline

(7b) and cyanoacetic acid (6) were reacted using a procedure similar

to the synthesis of 8a, aﬀording compound 8b as a white solid (70%

1

yield). H NMR (400 MHz, DMSO-d₆) δ 10.47 (s, 1H), 7.61−7.52

(m, 2H), 7.42−7.33 (m, 2H), 3.91 (s, 2H).

2-Cyano-N-phenylacetamide (8c). Aniline (7c) and cyanoacetic

acid (6) were reacted using a procedure similar to the synthesis of 8a,

1

aﬀording compound 8c as a white solid (68% yield). H NMR (400

MHz, DMSO-d₆) δ 10.28 (s, 1H), 7.56−7.49 (m, 2H), 7.32 (t, J = 7.9

Hz, 2H), 7.08 (tt, J = 7.4, 1.3 Hz, 1H), 3.88 (s, 2H).

2-Cyano-N-(4-ethylphenyl)acetamide (8d). 4-Ethylaniline (7d)

and cyanoacetic acid (6) were reacted using a procedure similar to the

synthesis of 8a, aﬀording compound 8d as a white solid (75% yield).

¹H NMR (400 MHz, DMSO-d₆) δ 10.28 (s, 1H), 7.52−7.40 (J = 8.2

Hz, 2H), 7.16 (d, J = 8.2 Hz, 2H), 3.88 (s, 2H), 2.55 (q, J = 7.6 Hz,

2H), 1.15 (t, J = 7.6 Hz, 3H).

EXPERIMENTAL SECTION

Chemistry. Unless otherwise noted, all solvents and reagents were

commercially available (Bide, J&K, Sinopharm, etc.) and used without

■

2-Cyano-N-(4-methoxyphenyl)acetamide (8e). 4-Methoxyaniline

(7e) and cyanoacetic acid (6) were reacted using a procedure similar

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to the synthesis of 8a, aﬀording compound 8e as a white solid (60%

using a procedure similar to the synthesis of 11a, aﬀording compound

11d as a white solid (60% yield). H NMR (400 MHz, DMSO-d₆) δ

1

yield). H NMR (400 MHz, DMSO-d₆) δ 10.16 (s, 1H), 7.50−7.41

(m, 2H), 6.95−6.86 (m, 2H), 3.85 (s, 2H), 3.72 (s, 3H).

7.93−7.80 (m, 2H), 7.69 (dd, J = 8.5, 2.0 Hz, 1H), 3.86 (s, 3H).

Methyl 2-Isothiocyanatobenzoate (11e). Methyl 2-aminoben-

zoate (9e) and thiophosgene (10) were reacted using a procedure

similar to the synthesis of 11a, aﬀording compound 11e as a colorless

oil (75% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.90 (dd, J = 7.9,

1.6 Hz, 1H), 7.70−7.59 (m, 1H), 7.53−7.40 (m, 2H), 3.85 (s, 3H).

Methyl 2-Isothiocyanato-4-methoxybenzoate (11f). Methyl 2-

amino-4-methoxybenzoate (9f) and thiophosgene (10) were reacted

using a procedure similar to the synthesis of 11a, aﬀording compound

2-Cyano-N-(3-methoxyphenyl)acetamide (8f). 3-Methoxyaniline

(7f) and cyanoacetic acid (6) were reacted using a procedure similar

to the synthesis of 8a, aﬀording compound 8f as a white solid (75%

1

yield). H NMR (400 MHz, DMSO-d₆) δ 10.29 (s, 1H), 7.26−7.18

(m, 2H), 7.07 (d, J = 8.1 Hz, 1H), 6.68 (dd, J = 8.2, 1.8 Hz, 1H), 3.89

(s, 2H), 3.73 (s, 3H).

2-Cyano-N-(2-methoxyphenyl)acetamide (8g). 2-Methoxyaniline

(7g) and cyanoacetic acid (6) were reacted using a procedure similar

to the synthesis of 8a, aﬀording compound 8g as a white solid (50%

yield). ¹H NMR (400 MHz, DMSO-d₆) δ 9.61 (s, 1H), 7.91 (dd, J =

8.0, 1.6 Hz, 1H), 7.17−7.02 (m, 2H), 6.93 (td, J = 7.6, 1.6 Hz, 1H),

4.00 (s, 2H), 3.84 (s, 3H).

2-Cyano-N-(2-ethylphenyl)acetamide (8h). 2-Ethylaniline (7h)

and cyanoacetic acid (6) were reacted using a procedure similar to the

synthesis of 8a, aﬀording compound 8h as a white solid (70% yield).

¹H NMR (400 MHz, DMSO-d₆) δ 9.67 (s, 1H), 7.49−7.29 (m, 1H),

7.25 (dt, J = 7.4, 3.2 Hz, 1H), 7.18 (hept, J = 5.0 Hz, 2H), 3.93 (s,

2H), 2.58 (q, J = 7.6 Hz, 2H), 1.12 (t, J = 7.6 Hz, 3H).

2-Cyano-N-(2,5-dimethoxyphenyl)acetamide (8i). 2,5-Dimethox-

yaniline (7i) and cyanoacetic acid (6) were reacted using a procedure

similar to the synthesis of 8a, aﬀording compound 8i as a white solid

(80% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 9.59 (s, 1H), 7.63 (d,

J = 3.0 Hz, 1H), 6.96 (d, J = 8.9 Hz, 1H), 6.65 (dd, J = 8.9, 3.1 Hz,

1H), 4.00 (s, 2H), 3.77 (s, 3H), 3.67 (s, 3H).

1

11f as a white solid (72% yield). H NMR (400 MHz, DMSO-d₆) δ

7.92 (d, J = 8.9 Hz, 1H), 7.18 (d, J = 2.6 Hz, 1H), 7.05 (dd, J = 8.9,

2.6 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H).

Methyl 5-Chloro-2-isothiocyanatobenzoate (11g). Methyl 2-

amino-5-chlorobenzoate (9g) and thiophosgene (10) were reacted

using a procedure similar to the synthesis of 11a, aﬀording compound

1

11g as a white solid (78% yield). H NMR (400 MHz, DMSO-d₆) δ

7.92 (d, J = 2.5 Hz, 1H), 7.76 (dd, J = 8.7, 2.5 Hz, 1H), 7.60 (d, J =

8.6 Hz, 1H), 3.88 (s, 3H).

Methyl 5-Bromo-2-isothiocyanatobenzoate (11h). Methyl 2-

amino-5-bromobenzoate (9h) and thiophosgene (10) were reacted

using a procedure similar to the synthesis of 11a, aﬀording compound

1

11h as a yellow oil (58% yield). H NMR (400 MHz, DMSO-d₆) δ

8.04 (d, J = 2.4 Hz, 1H), 7.88 (dd, J = 8.5, 2.4 Hz, 1H), 7.52 (d, J =

8.6 Hz, 1H), 3.87 (s, 3H).

Methyl 2-Isothiocyanato-5-methoxybenzoate (11i). Methyl 2-

amino-5-methoxybenzoate (9i) and thiophosgene (10) were reacted

using a procedure similar to the synthesis of 11a, aﬀording compound

11i as a yellow solid (68% yield). ¹H NMR (400 MHz, DMSO-d₆) δ

7.57−7.45 (m, 1H), 7.38 (d, J = 3.0 Hz, 1H), 7.23 (d, J = 8.5 Hz,

1H), 3.88 (s, 3H), 3.82 (s, 3H).

2-Cyano-N-(3,5-dichlorophenyl)acetamide (8j). 3,5-Dichloroani-

line (7j) and cyanoacetic acid (6) were reacted using a procedure

similar to the synthesis of 8a, aﬀording compound 8j as a white solid

1

(52% yield). H NMR (400 MHz, DMSO-d₆) δ 10.65 (s, 1H), 7.58

(d, J = 1.9 Hz, 2H), 7.32 (t, J = 1.9 Hz, 1H), 3.94 (s, 2H).

2-Cyano-N-(3,4-dichlorophenyl)acetamide (8k). 3,4-Dichloroani-

line (7k) and cyanoacetic acid (6) were reacted using a procedure

similar to the synthesis of 8a, aﬀording compound 8k as a white solid

Methyl 2-Isothiocyanato-4,5-dimethoxybenzoate (11j). Methyl

2-amino-4,5-dimethoxybenzoate (9j) and thiophosgene (10) were

reacted using a procedure similar to the synthesis of 11a, aﬀording

1

(60% yield). H NMR (400 MHz, DMSO-d₆) δ 10.73 (s, 1H), 7.91

compound 11j as a yellow solid (70% yield). H NMR (400 MHz,

(d, J = 2.4 Hz, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.45 (dd, J = 8.8, 2.4 Hz,

1H), 3.95 (s, 2H).

DMSO-d₆) δ 7.39 (s, 1H), 7.21 (s, 1H), 3.85 (s, 3H), 3.84 (s, 3H),

3.81 (s, 3H).

N-(3-Chloro-2-nitrophenyl)-2-cyanoacetamide (8l). 3-Chloro-2-

nitroaniline (7l) and cyanoacetic acid (6) were reacted using a

procedure similar to the synthesis of 8a, aﬀording compound 8l as a

white solid (60% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (s,

1H), 7.80−7.64 (m, 3H), 3.97 (s, 2H).

Methyl 4,5-Diﬂuoro-2-isothiocyanatobenzoate (11k). Methyl 2-

amino-4,5-diﬂuorobenzoate (9k) and thiophosgene (10) were reacted

using a procedure similar to the synthesis of 11a, aﬀording compound

11k as a yellow solid (60% yield). ¹H NMR (400 MHz, DMSO-d₆) δ

7.96 (dd, J = 10.9, 8.6 Hz, 1H), 7.86 (dd, J = 11.0, 7.1 Hz, 1H), 3.86

(s, 3H).

General Procedure for the Preparation of 11a−11k. Dimethyl 2-

Isothiocyanatoterephthalate (11a). To a solution of dimethyl 2-

aminoterephthalate (9a, 1.00 g, 4.78 mmol, 1.0 equiv) in dichloro-

methane (100 mL) was slowly added thiophosgene (10, 0.66 g, 5.74

mmol, 1.2 equiv). Then, the saturated sodium bicarbonate solution

(10 mL, excess) was poured into the above mixture solution. The

resulting mixture was stirred at room temperature overnight. Upon

completion of the reaction, the reaction solution was poured into

dichloromethane (100 mL), and the organic layer was washed with

water (3 × 50 mL) and brine (3 × 50 mL), dried over sodium sulfate,

and concentrated to give compound 11a (0.85 g, 71% yield) as a

General Procedure for the Preparation of 14a−14k, 15a−15r,

and 16a−16h. 5-Oxo-N-phenyl-1-thioxo-4,5-dihydro-1H-thiazolo-

[3,4-a]quinazoline-3-carboxamide (14a). To a solution of 2-cyano-

N-phenylacetamide (8c, 0.2 g, 1.25 mmol, 1.0 equiv) in DMF (8 mL)

were added methyl 2-isothiocyanatobenzoate (11e, 0.24 g, 1.25

mmol, 1.0 equiv) and sulfur (12, 0.04 g, 1.46 mmol, 1.5 equiv). Then,

triethylamine (0.25 g, 2.50 mmol, 2.0 equiv) was added to the above

mixture solution. The resulting mixture was stirred at 50 °C overnight.

Upon completion of the reaction, the resulting mixture was poured

into water acidiﬁed with acetic acid (20 mL, 3% solution) and a

yellow precipitate appeared. The obtained precipitate was ﬁltered oﬀ

and triple-crystallized from the mixture of dichloromethane/methanol

(1:5) to give the compound 14a (0.17 g, yield 50%) as a yellow solid.

mp >300 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.79 (s, 1H), 10.59

(d, J = 8.7 Hz, 1H), 10.10 (s, 1H), 8.38−8.13 (m, 1H), 7.91 (t, J = 8.1

Hz, 1H), 7.67 (dd, J = 19.4, 7.8 Hz, 3H), 7.36 (t, J = 7.7 Hz, 2H),

7.14 (t, J = 7.3 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 183.4,

159.6, 143.7, 139.0, 138.2, 134.3, 129.2, 128.5, 128.2, 124.9, 121.6,

117.9. HRMS (AP-ESI) m/z calcd for C₁₇H₁₀N₃O₂S₂[M − H]⁻,

352.0220; found, 352.0213. Retention time: 6.6 min, eluted with 56%

methanol/44% water (containing 0.1% triethylamine).

1

white solid. H NMR (400 MHz, DMSO-d₆) δ 8.08−8.02 (m, 1H),

7.96 (d, J = 7.2 Hz, 2H), 3.89 (s, 3H), 3.88 (s, 3H).

Methyl 4-Fluoro-2-isothiocyanatobenzoate (11b). Methyl 2-

amino-4-ﬂuorobenzoate (9b) and thiophosgene (10) were reacted

using a procedure similar to the synthesis of 11a, aﬀording compound

11b as a colorless oil (65% yield). ¹H NMR (400 MHz, DMSO-d₆) δ

8.02 (dd, J = 8.9, 6.3 Hz, 1H), 7.58 (dd, J = 9.4, 2.7 Hz, 1H), 7.45−

7.27 (m, 1H), 3.88 (s, 3H).

Methyl 4-Chloro-2-isothiocyanatobenzoate (11c). Methyl 2-

amino-4-chlorobenzoate (9c) and thiophosgene (10) were reacted

using a procedure similar to the synthesis of 11a, aﬀording compound

1

11c as a white solid (70% yield). H NMR (400 MHz, DMSO-d₆) δ

N-(4-Nitrophenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-

a]quinazoline-3-carboxamide (14b). 2-Cyano-N-(4-nitrophenyl)-

acetamide (8a), methyl 2-isothiocyanatobenzoate (11e), and sulfur

(12) were reacted using a procedure similar to the synthesis of 14a,

aﬀording compound 14b as a yellow solid (44% yield). mp 292−294

7.95 (d, J = 8.6 Hz, 1H), 7.78 (d, J = 2.1 Hz, 1H), 7.57 (dd, J = 8.5,

2.2 Hz, 1H), 3.88 (s, 3H).

Methyl 4-Bromo-2-isothiocyanatobenzoate (11d). Methyl 2-

amino-4-bromobenzoate (9d) and thiophosgene (10) were reacted

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1

°C. H NMR (400 MHz, DMSO-d₆) δ 10.57 (d, J = 8.4 Hz, 1H),

J = 8.8 Hz, 1H), 9.75 (s, 1H), 8.21 (dd, J = 7.8, 1.8 Hz, 1H), 7.91

(ddd, J = 8.9, 7.3, 1.8 Hz, 1H), 7.69 (t, J = 7.5 Hz, 1H), 7.49 (d, J =

7.6 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 7.02−

6.94 (m, 1H), 3.83 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 183.4,

139.0, 134.1, 128.4, 128.1, 120.7, 118.3, 117.9, 112.2, 56.1. HRMS

(AP-ESI) m/z calcd for C₁₈H₁₂N₃O₃S₂[M − H]⁻, 382.0326; found,

382.0320. Retention time: 5.1 min, eluted with 56% methanol/44%

water (containing 0.1% triethylamine).

10.44 (s, 1H), 8.20 (dd, J = 7.8, 1.8 Hz, 1H), 7.90 (ddd, J = 8.9, 7.3,

1.8 Hz, 1H), 7.77 (d, J = 1.9 Hz, 2H), 7.68 (t, J = 7.5 Hz, 1H), 7.33

(t, J = 1.9 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ 183.5, 159.6,

144.4, 140.8, 138.9, 134.5, 134.3, 128.5, 128.1, 123.7, 119.0, 117.8.

HRMS (AP-ESI) m/z calcd for C₁₇H₉N₄O₂S₂[M − H]⁻, 397.0071;

found, 397.0066. Retention time: 6.1 min, eluted with 70% methanol/

30% water (containing 0.1% triethylamine).

N-(4-Chlorophenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo-

[3,4-a]quinazoline-3-carboxamide (14c). N-(4-Chlorophenyl)-2-

cyanoacetamide (8b), methyl 2-isothiocyanatobenzoate (11e), and

sulfur (12) were reacted using a procedure similar to the synthesis of

14a, aﬀording compound 14c as a yellow solid (40% yield). mp 298−

300 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.72 (s, 1H), 10.59 (d, J

= 8.5 Hz, 1H), 10.21 (s, 1H), 8.21 (dd, J = 7.8, 1.8 Hz, 1H), 7.92

(ddd, J = 8.8, 7.2, 1.8 Hz, 1H), 7.70 (tt, J = 5.5, 2.1 Hz, 3H), 7.47−

7.37 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ 183.4, 159.6, 143.7,

138.9, 137.2, 134.3, 129.1, 128.5, 128.1, 122.9, 117.9. HRMS (AP-

ESI) m/z calcd for C₁₇H₉ClN₃O₂S₂[M − H]⁻, 385.9830; found,

385.9824. Retention time: 14.2 min, eluted with 56% methanol/44%

water (containing 0.1% triethylamine).

N-(2,5-Dimethoxyphenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-

thiazolo[3,4-a]quinazoline-3-carboxamide (14h). 2-Cyano-N-(2,5-

dimethoxyphenyl)acetamide (8i), methyl 2-isothiocyanatobenzoate

(11e), and sulfur (12) were reacted using a procedure similar to the

synthesis of 14a, aﬀording compound 14h as a yellow solid (54%

yield). mp 246−248 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.58 (d,

J = 8.7 Hz, 1H), 9.72 (s, 1H), 8.28−8.14 (m, 1H), 7.67 (t, J = 7.5 Hz,

1H), 7.18 (s, 1H), 7.00 (d, J = 9.0 Hz, 1H), 6.79 (dd, J = 9.3, 3.3 Hz,

1H), 3.75 (s, 3H), 3.70 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ

183.4, 159.8, 157.8, 153.2, 143.6, 139.0, 134.1, 128.4, 128.1, 126.4,

118.1, 117.8, 112.9, 56.6, 55.9. HRMS (AP-ESI) m/z calcd for

C₁₉H₁₄N₃O₄S₂[M − H]⁻, 412.0431; found, 412.0420. Retention

time: 6.2 min, eluted with 56% methanol/44% water (containing 0.1%

triethylamine).

N-(4-Ethylphenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-

a]quinazoline-3-carboxamide (14d). 2-Cyano-N-(4-ethylphenyl)-

acetamide (8d), methyl 2-isothiocyanatobenzoate (11e), and sulfur

(12) were reacted using a procedure similar to the synthesis of 14a,

aﬀording compound 14d as a yellow solid (45% yield). mp 298−300

N-(2-Ethylphenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo[3,4-

a]quinazoline-3-carboxamide (14i). 2-Cyano-N-(2-ethylphenyl)-

acetamide (8h), methyl 2-isothiocyanatobenzoate (11e), and sulfur

(12) were reacted using a procedure similar to the synthesis of 14a,

aﬀording compound 14i as a yellow solid (55% yield). mp 234−236

1

°C. H NMR (400 MHz, DMSO-d₆) δ 10.61 (d, J = 9.0 Hz, 1H),

1

10.07 (s, 1H), 8.22 (dd, J = 7.8, 1.8 Hz, 1H), 8.04−7.83 (m, 1H),

7.70 (t, J = 7.6 Hz, 1H), 7.56 (d, J = 8.2 Hz, 2H), 7.20 (d, J = 8.2 Hz,

2H), 2.58 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H). ¹³C NMR

(151 MHz, DMSO-d₆) δ 183.3, 159.5, 140.4, 139.0, 135.8, 134.3,

128.5, 128.4, 128.2, 121.7, 117.9. HRMS (AP-ESI) m/z calcd for

C₁₉H₁₄N₃O₂S₂[M − H]⁻, 380.0533; found, 380.0525. Retention

time: 11.3 min, eluted with 56% methanol/44% water (containing

0.1% triethylamine).

°C. H NMR (400 MHz, DMSO-d₆) δ 11.71 (s, 1H), 10.59 (d, J =

8.8 Hz, 1H), 9.96 (s, 1H), 8.21 (dd, J = 7.8, 1.8 Hz, 1H), 7.91 (t, J =

8.0 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.30 (d, J = 22.5 Hz, 4H), 2.61

(q, J = 7.6 Hz, 2H), 1.14 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 183.4, 138.9, 134.1, 129.2, 128.4, 128.2, 126.7, 118.4,

117.9, 24.4, 14.6. HRMS (AP-ESI) m/z calcd for C₁₉H₁₄N₃O₂S₂[M

− H]⁻, 380.0533; found, 380.0526. Retention time: 8.4 min, eluted

with 56% methanol/44% water (containing 0.1% triethylamine).

N-(3,5-Dichlorophenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo-

[3,4-a]quinazoline-3-carboxamide (14j). 2-Cyano-N-(3,5-

dichlorophenyl)acetamide (8j), methyl 2-isothiocyanatobenzoate

(11e), and sulfur (12) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 14j as a yellow solid (47%

N-(4-Methoxyphenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo-

[3,4-a]quinazoline-3-carboxamide (14e). 2-Cyano-N-(4-

methoxyphenyl)acetamide (8e), methyl 2-isothiocyanatobenzoate

(11e), and sulfur (12) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 14e as a yellow solid (43%

yield). mp 291−293 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.87 (s,

1H), 10.59 (d, J = 8.7 Hz, 1H), 9.98 (s, 1H), 8.21 (dd, J = 7.8, 1.8 Hz,

1H), 7.91 (ddd, J = 8.9, 7.2, 1.8 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H),

7.54 (d, J = 8.5 Hz, 2H), 6.98−6.83 (m, 2H), 3.74 (s, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 183.3, 159.4, 156.6, 143.2, 138.9, 134.3,

131.0, 128.5, 128.2, 123.3, 118.2, 117.9, 114.3, 55.6. HRMS (AP-ESI)

m/z calcd for C₁₈H₁₂N₃O₃S₂[M − H]⁻, 382.0326; found, 382.0324.

Retention time: 6.0 min, eluted with 56% methanol/44% water

(containing 0.1% triethylamine).

N-(3-Methoxyphenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo-

[3,4-a]quinazoline-3-carboxamide (14f). 2-Cyano-N-(3-

methoxyphenyl)acetamide (8f), methyl 2-isothiocyanatobenzoate

(11e), and sulfur (12) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 14f as a yellow solid (50%

yield). mp 283−285 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 11.82 (s,

1H), 10.60 (d, J = 8.7 Hz, 1H), 10.05 (s, 1H), 8.22 (dd, J = 7.7, 1.8

Hz, 1H), 7.92 (ddd, J = 8.9, 7.2, 1.8 Hz, 1H), 7.77−7.63 (m, 1H),

7.33 (d, J = 2.5 Hz, 1H), 7.29−7.23 (m, 2H), 6.71 (dq, J = 6.5, 4.0,

3.5 Hz, 1H), 3.76 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 183.4,

159.8, 159.6, 143.6, 139.4, 138.9, 134.3, 129.9, 128.5, 128.1, 117.9,

113.6, 110.4, 107.1, 55.5. HRMS (AP-ESI) m/z calcd for

C₁₈H₁₂N₃O₃S₂[M − H]⁻, 382.0326; found, 382.0315. Retention

time: 7.9 min, eluted with 56% methanol/44% water (containing 0.1%

triethylamine).

1

yield). mp >300 °C. H NMR (400 MHz, DMSO-d₆) δ 10.68 (s,

1H), 10.55 (d, J = 9.8 Hz, 1H), 8.21 (d, J = 8.7 Hz, 3H), 7.90 (dd, J =

18.5, 8.6 Hz, 3H), 7.67 (t, J = 7.5 Hz, 1H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 183.6, 159.7, 144.7, 143.1, 138.9, 134.3, 128.6, 128.1,

125.2, 120.6, 117.8. HRMS (AP-ESI) m/z calcd for C₁₇H₈Cl₂N₃O₂S₂

[M − H]⁻, 419.9440; found, 419.9441. Retention time: 15.1 min,

eluted with 56% methanol/44% water (containing 0.1% triethyl-

amine).

N-(3,4-Dichlorophenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo-

[3,4-a]quinazoline-3-carboxamide (14k). 2-Cyano-N-(3,4-

dichlorophenyl)acetamide (8k), methyl 2-isothiocyanatobenzoate

(11e), and sulfur (12) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 14k as a yellow solid (55%

yield). mp 290−292 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.58 (d,

J = 8.8 Hz, 1H), 10.37 (d, J = 5.4 Hz, 1H), 8.33−8.14 (m, 1H), 8.02

(d, J = 2.3 Hz, 1H), 7.93 (dt, J = 15.9, 7.9 Hz, 1H), 7.79−7.58 (m,

3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 183.5, 159.6, 138.9, 138.5,

134.3, 131.4, 131.1, 128.5, 128.1, 126.2, 122.3, 121.0, 117.8. HRMS

(AP-ESI) m/z calcd for C₁₇H₈Cl₂N₃O₂S₂[M − H]⁻, 419.9440;

found, 419.9436. Retention time: 14.9 min, eluted with 60%

methanol/40% water (containing 0.1% triethylamine).

N-(2,5-Dimethoxyphenyl)-8-methoxy-5-oxo-1-thioxo-4,5-dihy-

dro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15a). 2-Cyano-

N-(2,5-dimethoxyphenyl)acetamide (8i), methyl 2-isothiocyanato-4-

methoxybenzoate (11f), and sulfur (12) were reacted using a

procedure similar to the synthesis of 14a, aﬀording compound 15a

as a yellow solid (40% yield). mp 272−274 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.38 (s, 1H), 9.91 (s, 1H), 8.13 (d, J = 8.7 Hz, 1H),

7.26 (dd, J = 8.9, 2.5 Hz, 2H), 7.02 (d, J = 9.0 Hz, 1H), 6.79 (d, J =

8.9 Hz, 1H), 3.90 (s, 3H), 3.78 (s, 3H), 3.72 (s, 3H). ¹³C NMR (126

N-(2-Methoxyphenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-thiazolo-

[3,4-a]quinazoline-3-carboxamide (14g). 2-Cyano-N-(2-

methoxyphenyl)acetamide (8g), methyl 2-isothiocyanatobenzoate

(11e), and sulfur (12) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 14g as a yellow solid (48%

yield). mp 270−272 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.60 (d,

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MHz, DMSO-d₆) δ 183.5, 153.3, 140.4, 129.9, 114.5, 112.9, 103.3,

56.6, 56.6, 55.9. HRMS (AP-ESI) m/z calcd for C₂₀H₁₆N₃O₅S₂[M −

H]⁻, 442.0537; found, 442.0528. Retention time: 8.2 min, eluted with

56% methanol/44% water (containing 0.1% triethylamine).

calcd for C₂₀H₁₆N₃O₅S₂[M − H]⁻, 442.0537; found, 442.0530.

Retention time: 8.5 min, eluted with 56% methanol/44% water

(containing 0.1% triethylamine).

7-Bromo-N-(2,5-dimethoxyphenyl)-5-oxo-1-thioxo-4,5-dihydro-

1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15g). 2-Cyano-N-

(2,5-dimethoxyphenyl)acetamide (8i), methyl 5-bromo-2-isothiocya-

natobenzoate (11h), and sulfur (12) were reacted using a procedure

similar to the synthesis of 14a, aﬀording compound 15g as a yellow

solid (40% yield). mp 263−265 °C. ¹H NMR (400 MHz, DMSO-d₆)

δ 10.57 (d, J = 9.0 Hz, 1H), 10.06 (s, 1H), 8.23 (d, J = 2.7 Hz, 1H),

8.07 (dd, J = 8.9, 2.6 Hz, 1H), 7.28 (s, 1H), 6.98 (d, J = 8.9 Hz, 1H),

6.76 (d, J = 9.0 Hz, 1H), 3.76 (s, 3H), 3.70 (s, 3H). ¹³C NMR (126

MHz, pyridine-d₅) δ 183.9, 162.9, 159.3, 154.2, 147.1, 143.3, 138.6,

134.0, 130.0, 121.0, 120.1, 119.8, 112.0, 107.73, 96.7, 56.5, 55.3.

HRMS (AP-ESI) m/z calcd for C₁₉H₁₃BrN₃O₄S₂[M − H]⁻,

489.9536; found, 489.9534. Retention time: 19.8 min, eluted with

56% methanol/44% water (containing 0.1% triethylamine).

7-Chloro-N-(2,5-dimethoxyphenyl)-5-oxo-1-thioxo-4,5-dihydro-

1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15h). 2-Cyano-N-

(2,5-dimethoxyphenyl)acetamide (8i), methyl 5-chloro-2-isothiocya-

natobenzoate (11 g), and sulfur (12) were reacted using a procedure

similar to the synthesis of 14a, aﬀording compound 15h as a yellow

solid (46% yield). mp 256−258 °C. ¹H NMR (400 MHz, DMSO-d₆)

δ 10.66 (d, J = 9.3 Hz, 1H), 10.04 (s, 1H), 8.11 (d, J = 2.8 Hz, 1H),

7.96 (dd, J = 9.3, 2.8 Hz, 1H), 7.27 (s, 1H), 6.99 (d, J = 9.0 Hz, 1H),

6.84−6.70 (m, 1H), 3.77 (s, 3H), 3.72 (s, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 183.3, 159.6, 157.4, 153.3, 143.9, 137.7, 133.4, 132.3,

127.0, 126.7, 120.4, 119.8, 112.8, 111.1, 56.6, 55.9. HRMS (AP-ESI)

m/z calcd for C₁₉H₁₃ClN₃O₄S₂[M − H]⁻, 446.0041; found,

446.0034. Retention time: 15.7 min, eluted with 56% methanol/

44% water (containing 0.1% triethylamine).

8-Bromo-N-(2,5-dimethoxyphenyl)-5-oxo-1-thioxo-4,5-dihydro-

1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15b). 2-Cyano-N-

(2,5-dimethoxyphenyl)acetamide (8i), methyl 4-bromo-2-isothiocya-

natobenzoate (11d), and sulfur (12) were reacted using a procedure

similar to the synthesis of 14a, aﬀording compound 15b as a yellow

solid (45% yield). mp 292−294 °C. ¹H NMR (400 MHz, DMSO-d₆)

δ 10.99 (s, 1H), 10.31 (s, 1H), 8.11 (d, J = 8.3 Hz, 1H), 7.88 (dd, J =

8.4, 1.9 Hz, 1H), 7.37 (s, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.75 (d, J =

8.8 Hz, 1H), 3.79 (s, 3H), 3.71 (s, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 183.6, 153.3, 139.6, 131.1, 129.7, 120.2, 117.9, 112.8,

56.7, 55.9. HRMS (AP-ESI) m/z calcd for C₁₉H₁₃BrN₃O₄S₂[M −

H]⁻, 489.9536; found, 489.9543. Retention time: 17.0 min, eluted

with 56% methanol/44% water (containing 0.1% triethylamine).

8-Chloro-N-(2,5-dimethoxyphenyl)-5-oxo-1-thioxo-4,5-dihydro-

1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15c). 2-Cyano-N-

(2,5-dimethoxyphenyl)acetamide (8i), methyl 4-chloro-2-isothiocya-

natobenzoate (11c), and sulfur (12) were reacted using a procedure

similar to the synthesis of 14a, aﬀording compound 15c as a yellow

solid (47% yield). mp 283−285 °C. ¹H NMR (400 MHz, DMSO-d₆)

δ 10.85 (s, 1H), 10.06 (s, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.76 (dd, J =

8.4, 2.1 Hz, 1H), 7.30 (s, 1H), 7.01 (d, J = 9.0 Hz, 1H), 6.83−6.74

(m, 1H), 3.79 (s, 3H), 3.73 (s, 3H). ¹³C NMR (126 MHz, DMSO-

d₆) δ 183.6, 153.3, 139.6, 137.9, 129.7, 128.3, 117.4, 112.8, 56.6, 55.9.

HRMS (AP-ESI) m/z calcd for C₁₉H₁₃ClN₃O₄S₂[M − H]⁻,

446.0041; found, 446.0044. Retention time: 14.4 min, eluted with

56% methanol/44% water (containing 0.1% triethylamine).

N-(2,5-Dimethoxyphenyl)-8-ﬂuoro-5-oxo-1-thioxo-4,5-dihydro-

1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15d). 2-Cyano-N-

(2,5-dimethoxyphenyl)acetamide (8i), methyl 4-ﬂuoro-2-isothiocya-

natobenzoate (11b), and sulfur (12) were reacted using a procedure

similar to the synthesis of 14a, aﬀording compound 15d as a yellow

solid (52% yield). mp 255−277 °C. ¹H NMR (400 MHz, DMSO-d₆)

δ 10.59 (d, J = 14.4 Hz, 1H), 9.99 (s, 1H), 8.27 (dd, J = 8.8, 6.6 Hz,

1H), 7.57 (td, J = 9.3, 8.4, 2.5 Hz, 1H), 7.25 (s, 1H), 7.00 (d, J = 9.0

Hz, 1H), 6.91−6.70 (m, 1H), 3.77 (s, 3H), 3.72 (s, 3H). ¹³C NMR

(151 MHz, DMSO-d₆) δ 183.7, 164.9, 163.2, 153.2, 144.3, 140.3,

130.9, 130.9, 115.8, 115.7, 115.3, 112.8, 105.3, 105.1, 56.6, 55.9.

HRMS (AP-ESI) m/z calcd for C₁₉H₁₃FN₃O₄S₂[M − H]⁻,

430.0337; found, 430.0333. Retention time: 9.3 min, eluted with

56% methanol/44% water (containing 0.1% triethylamine).

N-(2,5-Dimethoxyphenyl)-7,8-diﬂuoro-5-oxo-1-thioxo-4,5-dihy-

dro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15i). 2-Cyano-

N-(2,5-dimethoxyphenyl)acetamide (8i), methyl 4,5-diﬂuoro-2-iso-

thiocyanatobenzoate (11k), and sulfur (12) were reacted using a

procedure similar to the synthesis of 14a, aﬀording compound 15i as

1

a yellow solid (44% yield). mp 254−256 °C. H NMR (400 MHz,

DMSO-d₆) δ 10.88 (dd, J = 14.1, 7.1 Hz, 1H), 10.52 (s, 1H), 8.13 (t,

J = 9.6 Hz, 1H), 7.44 (s, 1H), 6.96 (d, J = 8.9 Hz, 1H), 6.70 (d, J =

8.9 Hz, 1H), 3.78 (s, 3H), 3.69 (s, 3H). ¹³C NMR (126 MHz,

pyridine-d₅) δ 183.8, 162.7, 159.4, 154.3, 148.1, 143.3, 130.3, 117.8,

115.6, 112.2, 107.9, 107.4, 96.0, 56.7, 55.2. HRMS (AP-ESI) m/z

calcd for C₁₉H₁₂F₂N₃O₄S₂[M − H]⁻, 448.0243; found, 448.0249.

Retention time: 15.5 min, eluted with 56% methanol/44% water

(containing 0.1% triethylamine).

Methyl 3-((2,5-Dimethoxyphenyl)carbamoyl)-5-oxo-1-thioxo-

4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-8-carboxylate (15e). 2-

Cyano-N-(2,5-dimethoxyphenyl)acetamide (8i), dimethyl 2-isothio-

cyanatoterephthalate (11a), and sulfur (12) were reacted using a

procedure similar to the synthesis of 14a, aﬀording compound 15e as

N-(2,5-Dimethoxyphenyl)-7,8-dimethoxy-5-oxo-1-thioxo-4,5-di-

hydro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15j). 2-

Cyano-N-(2,5-dimethoxyphenyl)acetamide (8i), methyl 2-isothiocya-

nato-4,5-dimethoxybenzoate (11j), and sulfur (12) were reacted

using a procedure similar to the synthesis of 14a, aﬀording compound

1

a yellow solid (60% yield). mp 271−273 °C. H NMR (400 MHz,

DMSO-d₆) δ 11.31 (s, 1H), 10.36 (s, 1H), 8.29 (d, J = 8.1 Hz, 1H),

8.15 (d, J = 8.2 Hz, 1H), 7.39 (s, 1H), 6.98 (d, J = 8.9 Hz, 1H), 6.73

(d, J = 8.5 Hz, 1H), 3.92 (s, 3H), 3.78 (s, 3H), 3.70 (s, 3H). ¹³C

NMR (126 MHz, pyridine-d₅) δ 185.7, 167.4, 164.9, 160.8, 155.7,

151.4, 151.3, 151.1, 150.9, 148.7, 144.9, 141.0, 134.1, 131.4, 129.1,

123.6, 120.6, 113.5, 109.3, 58.0, 56.8, 53.8. HRMS (AP-ESI) m/z

calcd for C₂₁H₁₆Cl₂N₃O₆S₂[M − H]⁻, 470.0486; found, 470.0496.

Retention time: 9.4 min, eluted with 55% methanol/45% water

(containing 0.1% triethylamine).

1

15j as a yellow solid (45% yield). mp 286−288 °C. H NMR (400

MHz, DMSO-d₆) δ 10.51 (s, 1H), 9.91 (s, 1H), 7.58 (s, 1H), 7.27 (s,

1H), 7.02 (d, J = 9.0 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 3.91 (s, 3H),

3.89 (s, 3H), 3.79 (s, 3H), 3.72 (s, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 182.1, 153.3, 148.0, 134.0, 112.9, 108.5, 102.0, 56.8,

56.6, 56.3, 55.9. HRMS (AP-ESI) m/z calcd for C₂₁H₁₈N₃O₆S₂[M −

H]⁻, 472.0643; found, 472.0640. Retention time: 5.7 min, eluted with

56% methanol/44% water (containing 0.1% triethylamine).

8-Chloro-N-(4-nitrophenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-

thiazolo[3,4-a]quinazoline-3-carboxamide (15k). 2-Cyano-N-(4-

nitrophenyl)acetamide (8a), methyl 4-chloro-2-isothiocyanatoben-

zoate (11c), and sulfur (12) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 15k as a yellow solid (47%

yield). mp 296−297 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 12.35 (s,

1H), 10.88 (d, J = 2.1 Hz, 1H), 8.28−8.22 (m, 2H), 8.19 (d, J = 8.4

Hz, 1H), 7.87−7.80 (m, 2H), 7.67 (dd, J = 8.4, 2.1 Hz, 1H). ¹³C

NMR (101 MHz, pyridine-d₅) δ 183.9, 164.2, 159.5, 145.4, 141.4,

139.0, 128.9, 127.1, 124.5, 118.9, 116.6, 93.6. HRMS (AP-ESI) m/z

calcd for C₁₇H₉ClN₄O₄S₂[M − H]⁻, 430.9681; found, 430.9695.

N-(2,5-Dimethoxyphenyl)-7-methoxy-5-oxo-1-thioxo-4,5-dihy-

dro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15f). 2-Cyano-

N-(2,5-dimethoxyphenyl)acetamide (8i), methyl 2-isothiocyanato-5-

methoxybenzoate (11i), and sulfur (12) were reacted using a

procedure similar to the synthesis of 14a, aﬀording compound 15f

as a yellow solid (42% yield). mp 250−252 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 10.57 (d, J = 9.5 Hz, 1H), 9.86 (s, 1H), 7.63 (d, J = 3.3

Hz, 1H), 7.47 (dd, J = 9.5, 3.2 Hz, 1H), 7.24 (s, 1H), 7.02 (d, J = 9.0

Hz, 1H), 6.89−6.70 (m, 1H), 3.91 (s, 3H), 3.78 (s, 3H), 3.72 (s, 3H).

¹³C NMR (126 MHz, DMSO-d₆) δ 182.1, 158.2, 153.3, 132.7, 120.1,

120.0, 119.9, 112.9, 110.8, 56.6, 56.2, 55.9. HRMS (AP-ESI) m/z

9231

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Retention time: 1.0 min, eluted with 56% methanol/44% water

(containing 0.1% triethylamine).

Chloro-2-nitrophenyl)-2-cyanoacetamide (8l), methyl 4-chloro-2-

isothiocyanatobenzoate (11c), and sulfur (12) were reacted using a

procedure similar to the synthesis of 14a, aﬀording compound 15q as

Methyl 3-((4-Nitrophenyl)carbamoyl)-5-oxo-1-thioxo-4,5-dihy-

dro-1H-thiazolo[3,4-a]quinazoline-8-carboxylate (15l). 2-Cyano-

N-(4-nitrophenyl)acetamide (8a), dimethyl 2-isothiocyanatotereph-

thalate (11a), and sulfur (12) were reacted using a procedure similar

to the synthesis of 14a, aﬀording compound 15l as a yellow solid

1

a yellow solid (54% yield). mp 238−240 °C. H NMR (400 MHz,

DMSO-d₆) δ 11.41 (s, 1H), 10.80 (s, 1H) , 8.17 (d, J = 8.4 Hz, 1H),

7.72 (dd, J = 22.4, 8.2 Hz, 2H), 7.57 (dq,J = 18.2, 9.9, 8.3 Hz, 2H).

¹³C NMR (101 MHz, DMSO-d₆) δ 183.6, 159.7, 144.5, 139.1, 138.0,

1

(44% yield). mp 274−275 °C. H NMR (400 MHz, DMSO-d₆) δ

132.5, 130.5, 129.5, 128.6, 128.2, 127.3, 124.9, 117.0. HRMS (AP-

ESI) m/z calcd for C₁₇H₈Cl₂N₄O₄S₂[M − H]⁻, 464.9291; found,

464.9296. Retention time: 5.8 min, eluted with 56% methanol/44%

water (containing 0.1% triethylamine).

12.59 (s, 1H), 11.39 (s, 1H), 8.28 (dd, J = 14.8, 8.4 Hz, 3H), 8.12 (d,

J = 8.2 Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 3.92 (s, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 183.4, 165.5, 162.2, 158.8, 149.9, 145.5,

141.4, 138.6, 131.2, 127.8, 127.3, 125.1, 122.6, 118.3, 118.2, 91.4,

52.6. HRMS (AP-ESI) m/z calcd for C₁₉H₁₂N₄O₆S₂[M − H]⁻,

455.0125; found, 455.0121. Retention time: 6.1 min, eluted with 56%

methanol/44% water (containing 0.1% triethylamine).

Methyl 3-((3-Chloro-2-nitrophenyl)carbamoyl)-5-oxo-1-thioxo-

4,5-dihydro-1H-thiazolo[3,4-a]quinazoline-8-carboxylate (15r). N-

(3-Chloro-2-nitrophenyl)-2-cyanoacetamide (8l), dimethyl 2-isothio-

cyanatoterephthalate (11a), and sulfur (12) were reacted using a

procedure similar to the synthesis of 14a, aﬀording compound 15r as

8-Chloro-N-(3,5-dichlorophenyl)-5-oxo-1-thioxo-4,5-dihydro-

1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15m). 2-Cyano-N-

(3,5-dichlorophenyl)acetamide (8j), methyl 4-chloro-2-isothiocyana-

tobenzoate (11c), and sulfur (12) were reacted using a procedure

similar to the synthesis of 14a, aﬀording compound 15m as a brown

1

a yellow solid (17% yield). mp 235−237 °C. H NMR (400 MHz,

DMSO-d₆) δ 11.20 (d, J = 1.5 Hz, 1H), 10.73 (s, 1H), 8.30 (d, J = 8.1

Hz, 1H), 8.17 (dd, J = 8.1, 1.5 Hz, 1H), 7.80−7.65 (m, 2H), 7.59 (dd,

J = 6.7, 2.7 Hz, 1H), 3.94 (s, 3H). ¹³C NMR (125 MHz, DMSO-d₆) δ

183.9, 165.4, 160.3, 145.2, 144.6, 138.9, 134.1, 133.0, 131.0, 129.2,

128.8, 128.6, 128.0, 125.4, 121.8, 118.7, 53.5. HRMS (AP-ESI) m/z

calcd for C₁₉H₁₁ClN₄O₆S₂[M − H]⁻, 488.9736; found, 488.9740.

Retention time: 3.9 min, eluted with 56% methanol/44% water

(containing 0.1% triethylamine).

1

solid (19% yield). mp >300 °C. H NMR (400 MHz, DMSO-d₆) δ

12.04 (s, 1H), 10.84 (s, 1H), 8.14 (d, J = 8.5 Hz, 1H), 7.61 (t, J =

10.7 Hz, 3H), 7.21 (s, 1H). ¹³C NMR (125 MHz, pyridine-d₅) δ

184.7, 163.1, 159.5, 148.2, 141.6, 140.1, 134.7, 129.3, 127.4, 117.8,

117.6, 117.6, 94.3. HRMS (AP-ESI) m/z calcd for C₁₇H₈Cl₃N₃O₂S₂

[M − H]⁻, 453.9051; found, 453.9054. Retention time: 23.2 min,

eluted with 56% methanol/44% water (containing 0.1% triethyl-

amine).

8-Chloro-N-(2-ethylphenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-

thiazolo[3,4-a]quinazoline-3-carboxamide (16a). 2-Cyano-N-(2-

ethylphenyl)acetamide (8h), methyl 4-chloro-2-isothiocyanatoben-

zoate (11c), and sulfur (12) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 16a as a yellow solid (53%

yield). mp 259−261 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.78 (s,

1H), 10.01 (s, 1H), 8.17 (d, J = 8.4 Hz, 1H), 7.75 (dd, J = 8.4, 2.0 Hz,

1H), 7.27 (dd, J = 19.4, 9.1 Hz, 4H), 2.59 (q, J = 7.5 Hz, 2H), 1.12 (t,

J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, pyridine-d₅) δ 184.1, 150.0,

153.0, 146.7, 140.0, 137.2, 135.5, 129.1, 128.6, 126.4, 124.9, 123.5,

117.6, 25.1, 14.3. HRMS (AP-ESI) m/z calcd for C₁₉H₁₃ClN₃O₂S₂

[M − H]⁻, 414.0143; found, 414.0136. Retention time: 17.8 min,

eluted with 56% methanol/44% water (containing 0.1% triethyl-

amine).

7-Bromo-N-(3,5-dichlorophenyl)-5-oxo-1-thioxo-4,5-dihydro-

1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15n). 2-Cyano-N-

(3,5-dichlorophenyl)acetamide (8j), methyl 5-bromo-2-isothiocyana-

tobenzoate (11h), and sulfur (12) were reacted using a procedure

similar to the synthesis of 14a, aﬀording compound 15n as a yellow

solid (47% yield). mp 271−273 °C. ¹H NMR (400 MHz, DMSO-d₆)

δ 10.81 (s, 1H), 10.58 (d, J = 9.2 Hz, 1H), 8.25 (d, J = 2.6 Hz, 1H),

8.08 (dd, J = 9.2, 2.6 Hz, 1H), 7.92 (s, 1H), 7.75 (d, J = 1.9 Hz, 2H),

7.34 (t, J = 1.9 Hz, 1H). ¹³C NMR (126 MHz, pyridine-d₅) δ 184.1,

162.3, 159.4, 148.7, 148.3, 141.6, 138.3, 133.3, 130.0, 121.8, 121.5,

119.8, 119.6, 117.2. HRMS (AP-ESI) m/z calcd for

C₁₇H₇BrCl₂N₃O₂S₂[M − H]⁻, 497.8546; found, 497.8551. Retention

time: 12.8 min, eluted with 56% methanol/44% water (containing

0.1% triethylamine).

Methyl 3-((3,5-Dichlorophenyl)carbamoyl)-5-oxo-1-thioxo-4,5-

dihydro-1H-thiazolo[3,4-a]quinazoline-8-carboxylate (15o). 2-

Cyano-N-(3,5-dichlorophenyl)acetamide (8j), dimethyl 2-isothiocya-

natoterephthalate (11a), and sulfur (12) were reacted using a

procedure similar to the synthesis of 14a, aﬀording compound 15o as

a yellow solid (16% yield). mp >300 °C. ¹H NMR (600 MHz,

DMSO-d₆) δ 11.49 (s, 1H), 11.27 (s, 1H), 8.24 (d, J = 8.1 Hz, 1H),

8.09 (dd, J = 8.1, 1.5 Hz, 1H), 7.63 (s, 2H), 7.22 (s, 1H), 3.91 (s,

3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 181.4, 169.8, 164.9, 164.6,

142.4, 138.9, 133.1, 127.4, 126.2, 121.9, 120.2, 118.7, 116.5, 49.1.

HRMS (AP-ESI) m/z calcd for C₁₉H₁₀Cl₂N₃O₄S₂[M − H]⁻,

477.9495; found, 477.9500. Retention time: 15.2 min, eluted with

56% methanol/44% water (containing 0.1% triethylamine).

Methyl 3-((2-Ethylphenyl)carbamoyl)-5-oxo-1-thioxo-4,5-dihy-

dro-1H-thiazolo[3,4-a]quinazoline-8-carboxylate (16b). 2-Cyano-

N-(2-ethylphenyl)acetamide (8h), dimethyl 2-isothiocyanatotereph-

thalate (11a), and sulfur (12) were reacted using a procedure similar

to the synthesis of 14a, aﬀording compound 16b as a yellow solid

1

(49% yield). mp 246−248 °C. H NMR (400 MHz, DMSO-d₆) δ

11.24 (s, 1H), 10.05 (s, 1H), 8.28 (d, J = 8.1 Hz, 1H), 8.15 (dd, J =

8.1, 1.5 Hz, 1H), 7.41−7.03 (m, 5H), 3.92 (s, 3H), 2.61 (t, J = 7.5 Hz,

2H), 1.13 (t, J = 7.5 Hz, 3H). ¹³C NMR (126 MHz, pyridine-d₅) δ

185.6, 167.3, 161.4, 151.4, 148.3, 140.9, 137.0, 134.3, 130.2, 129.3,

129.2, 127.9, 125.0, 123.7, 120.7, 120.6, 53.8, 26.6, 15.9. HRMS (AP-

ESI) m/z calcd for C₂₁H₁₆N₃O₄S₂[M − H]⁻, 438.0588; found,

438.0590. Retention time: 10.6 min, eluted with 56% methanol/44%

water (containing 0.1% triethylamine).

7-Bromo-N-(2-ethylphenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-

thiazolo[3,4-a]quinazoline-3-carboxamide (16c). 2-Cyano-N-(2-

ethylphenyl)acetamide (8h), methyl 5-bromo-2-isothiocyanatoben-

zoate (11h), and sulfur (12) were reacted using a procedure similar to

the synthesis of 14a, aﬀording compound 16c as a yellow solid (40%

yield). mp 253−255 °C. ¹H NMR (400 MHz, DMSO-d₆) δ 10.53 (d,

J = 10.1 Hz, 1H), 10.01 (s, 1H), 8.27−8.17 (m, 1H), 8.14−8.02 (m,

1H), 7.28 (d, J = 20.7 Hz, 4H), 2.59 (q, J = 7.6 Hz, 2H), 1.12 (t, J =

7.6 Hz, 3H). ¹³C NMR (126 MHz, pyridine-d₅) δ 183.8, 159.9, 150.0,

149.9, 146.8, 138.5, 135.5, 135.4, 134.1, 130.1, 128.7, 126.4, 123.5,

121.1, 120.2, 119.8, 25.1, 14.3. HRMS (AP-ESI) m/z calcd for

C₁₉H₁₃BrN₃O₂S₂[M − H]⁻, 457.9638; found, 457.9645. Retention

time: 25.3 min, eluted with 56% methanol/44% water (containing

0.1% triethylamine).

N-(3,5-Dichlorophenyl)-7,8-dimethoxy-5-oxo-1-thioxo-4,5-dihy-

dro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15p). 2-Cyano-

N-(3,5-dichlorophenyl)acetamide (8j), methyl 2-isothiocyanato-4,5-

dimethoxybenzoate (11j), and sulfur (12) were reacted using a

procedure similar to the synthesis of 14a, aﬀording compound 15p as

1

a yellow solid (23% yield). mp 293−294 °C. H NMR (600 MHz,

pyridine-d₅) δ 11.36 (s, 1H), 10.89 (s, 1H), 7.97 (s, 1H), 7.59 (s,

2H), 7.11 (s, 1H), 3.98 (s, 3H), 3.87 (s, 3H). ¹³C NMR (151 MHz,

pyridine-d₅) δ 182.9, 162.2, 159.4, 152.6, 148.2, 146.1, 141.2, 134.7,

118.3, 110.8, 108.2, 101.9, 94.5, 56.3, 55.6. HRMS (AP-ESI) m/z

calcd for C₁₉H₁₃Cl₂N₃O₄S₂[M − H]⁻, 479.9652; found, 479.9666.

Retention time: 11.0 min, eluted with 56% methanol/44% water

(containing 0.1% triethylamine).

N-(2-Ethylphenyl)-7,8-diﬂuoro-5-oxo-1-thioxo-4,5-dihydro-1H-

thiazolo[3,4-a]quinazoline-3-carboxamide (16d). 2-Cyano-N-(2-

ethylphenyl)acetamide (8h), methyl 4,5-diﬂuoro-2-isothiocyanato-

8-Chloro-N-(3-chloro-2-nitrophenyl)-5-oxo-1-thioxo-4,5-dihy-

dro-1H-thiazolo[3,4-a]quinazoline-3-carboxamide (15q). N-(3-

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benzoate (11k), and sulfur (12) were reacted using a procedure

similar to the synthesis of 14a, aﬀording compound 16d as a yellow

solid (45% yield). mp 247−249 °C. ¹H NMR (400 MHz, DMSO-d₆)

δ 10.80 (s, 1H), 10.18 (s, 1H), 8.16 (t, J = 9.5 Hz, 1H), 7.37 (s, 1H),

7.30 (s, 1H), 7.26−7.07 (m, 2H), 2.61 (q, J = 7.5 Hz, 2H), 1.13 (t, J =

7.5 Hz, 3H). ¹³C NMR (126 MHz, pyridine-d₅) δ 183.7, 161.6, 159.8,

137.2, 136.0, 128.7, 128.7, 126.4, 124.1, 118.0, 115.6, 108.0, 107.7,

25.3, 14.4. HRMS (AP-ESI) m/z calcd for C₁₉H₁₂F₂N₃O₂S₂[M −

H]⁻, 416.0344; found, 416.0345. Retention time: 21.9 min, eluted

with 56% methanol/44% water (containing 0.1% triethylamine).

Methyl 3-((4-Chlorophenyl)carbamoyl)-5-oxo-1-thioxo-4,5-dihy-

dro-1H-thiazolo[3,4-a]quinazoline-8-carboxylate (16e). N-(4-

Chlorophenyl)-2-cyanoacetamide (8b), dimethyl 2-isothiocyanatoter-

ephthalate (11a), and sulfur (12) were reacted using a procedure

similar to the synthesis of 14a, aﬀording compound 16e as a yellow

reacted using a procedure similar to the synthesis of 19a, aﬀording

compound 19b as a yellow solid (40% yield). H NMR (400 MHz,

DMSO-d₆) δ 7.86 (s, 1H), 7.37−7.18 (m, 3H), 7.12 (d, J = 8.1 Hz,

1H), 6.67 (d, J = 3.1 Hz, 1H), 6.45 (s, 2H), 6.37 (d, J = 8.9 Hz, 1H),

6.11 (dd, J = 9.0, 3.0 Hz, 1H), 3.16 (s, 3H), 3.08 (s, 3H).

4-Amino-N-(2,5-dimethoxyphenyl)-2-thioxo-3-(4-

(triﬂuoromethyl)phenyl)-2,3-dihydrothiazole-5-carboxamide (19c).

2-Cyano-N-(2,5-dimethoxyphenyl)acetamide (8i), 1-isothiocyanato-

4-(triﬂuoromethyl)benzene (18c), and sulfur (12) were reacted using

a procedure similar to the synthesis of 19a, aﬀording compound 19c

as a light yellow solid (60% yield). ¹H NMR (400 MHz, DMSO-d₆) δ

8.49 (s, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.1 Hz, 2H), 7.28

(d, J = 3.1 Hz, 1H), 7.06 (s, 2H), 6.99 (d, J = 9.0 Hz, 1H), 6.72 (dd, J

= 9.0, 3.1 Hz, 1H), 3.78 (s, 3H), 3.70 (s, 3H).

1

4-Amino-3-(4-chlorophenyl)-N-(2,5-dimethoxyphenyl)-2-thioxo-

2,3-dihydrothiazole-5-carboxamide (19d). 2-Cyano-N-(2,5-

dimethoxyphenyl)acetamide (8i), 1-chloro-4-isothiocyanatobenzene

(18d), and sulfur (12) were reacted using a procedure similar to the

synthesis of 19a, aﬀording compound 19d as a yellow solid (55%

1

solid (35% yield). mp >300 °C. H NMR (400 MHz, DMSO-d₆) δ

11.32 (s, 1H), 10.57 (s, 1H), 8.33 (d, J = 8.1 Hz, 1H), 8.19 (d, J = 8.1

Hz, 1H), 7.70 (d, J = 8.6 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 3.95 (s,

3H). ¹³C NMR (126 MHz, pyridine-d₅) δ 184.3, 165.9, 162.3, 159.6,

139.4, 138.4, 134.9, 132.6, 128.7, 128.0, 127.6, 121.2, 119.2, 119.1,

52.2. HRMS (AP-ESI) m/z calcd for C₁₉H₁₁ClN₃O₄S₂[M − H]⁻,

443.9885; found, 443.9886. Retention time: 10.5 min, eluted with

60% methanol/40% water (containing 0.1% triethylamine).

1

yield). H NMR (400 MHz, DMSO-d₆) δ 8.45 (s, 1H), 7.72−7.63

(m, 2H), 7.49−7.40 (m, 2H), 7.28 (d, J = 3.1 Hz, 1H), 7.02 (s, 2H),

6.98 (d, J = 9.0 Hz, 1H), 6.72 (dd, J = 8.9, 3.1 Hz, 1H), 3.77 (s, 3H),

3.69 (s, 3H).

4-Amino-N-(2,5-dimethoxyphenyl)-3-(3-ﬂuorophenyl)-2-thioxo-

2,3-dihydrothiazole-5-carboxamide (19e). 2-Cyano-N-(2,5-

dimethoxyphenyl)acetamide (8i), 1-ﬂuoro-3-isothiocyanatobenzene

(18e), and sulfur (12) were reacted using a procedure similar to the

synthesis of 19a, aﬀording compound 19e as a yellow solid (50%

Methyl 3-((3,4-Dichlorophenyl)carbamoyl)-5-oxo-1-thioxo-

3,3a,4,5-tetrahydro-1H-thiazolo[3,4-a]quinazoline-8-carboxylate

(16f). 2-Cyano-N-(3,4-dichlorophenyl)acetamide (8k), dimethyl 2-

isothiocyanatoterephthalate (11a), and sulfur (12) were reacted using

a procedure similar to the synthesis of 14a, aﬀording compound 16f

as a yellow solid (35% yield). mp 288−290 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 13.29 (s, 1H), 8.07 (d, J = 8.2 Hz, 1H), 8.01 (s, 1H),

7.89−7.80 (m, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 2.3 Hz,

1H), 7.37 (dd, J = 8.5, 2.3 Hz, 1H), 3.91 (s, 3H). ¹³C NMR (126

MHz, pyridine-d₅) δ 178.7, 166.7, 161.2, 141.8, 140.9, 137.8, 134.3,

134.0, 133.2, 132.8, 131.2, 130.2, 125.7, 121.1, 118.2, 54.0. HRMS

(AP-ESI) m/z calcd for C₁₉H₁₀Cl₂N₃O₄S₂[M − H]⁻, 477.9495;

found, 477.9541. Retention time: 3.4 min, eluted with 56% methanol/

44% water (containing 0.1% triethylamine).

8-Bromo-N-(2-methoxyphenyl)-5-oxo-1-thioxo-4,5-dihydro-1H-

thiazolo[3,4-a]quinazoline-3-carboxamide (16g). 2-Cyano-N-(2-

methoxyphenyl)acetamide (8g), methyl 4-bromo-2-isothiocyanato-

benzoate (11d), and sulfur (12) were reacted using a procedure

similar to the synthesis of 14a, aﬀording compound 16g as a yellow

solid (45% yield). mp 246−248 °C. ¹H NMR (400 MHz, DMSO-d₆)

δ 10.58 (d, J = 9.2 Hz, 1H), 10.02 (s, 1H), 8.24 (d, J = 2.6 Hz, 1H),

8.09 (dd, J = 9.2, 2.6 Hz, 1H), 7.57 (s, 1H), 7.24 (s, 1H), 7.10 (d, J =

8.1 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 3.83(s, 3H). ¹³C NMR (126

MHz, DMSO-d₆) δ 183.3, 138.2, 136.3, 130.0, 120.7, 120.7, 120.5,

120.0, 112.1, 56.1. HRMS (AP-ESI) m/z calcd for C₁₈H₁₁BrN₃O₃S₂

[M − H]⁻, 459.9431; found, 459.9431. Retention time: 8.6 min,

eluted with 56% methanol/44% water (containing 0.1% triethyl-

amine).

General Procedure for the Preparation of 19a−19f. 4-Amino-3-

(3-cyanophenyl)-N-(2,5-dimethoxyphenyl)-2-thioxo-2,3-dihydro-

thiazole-5-carboxamide (19a). To a solution of 2-cyano-N-(2,5-

dimethoxyphenyl)acetamide (8i, 0.41 g, 1.87 mmol, 1.0 equiv) in

EtOH (20 mL) were added 3-isothiocyanatobenzonitrile (18a, 0.30 g,

1.87 mmol, 1.0 equiv) and sulfur (12, 0.9 g, 2.81 mmol, 1.5 equiv).

Then, triethylamine (0.38 g, 3.75 mmol, 2.0 equiv) was added to the

above mixture solution. The resulting mixture was stirred at 50 °C

overnight. Upon completion of the reaction, a yellow precipitate

appeared. The obtained precipitate was ﬁltered oﬀ and triple-

crystallized from the mixture of ethyl acetate /petroleum ether

(1:2) to give the compound 19a (0.40 g, 50% yield) as a yellow solid.

¹H NMR (400 MHz, DMSO-d₆) δ 8.46 (s, 1H), 8.05 (dp, J = 3.4, 1.7

1

yield). H NMR (400 MHz, DMSO-d₆) δ 8.47 (s, 1H), 7.69 (q, J =

7.8 Hz, 1H), 7.53−7.42 (m, 2H), 7.35−7.25 (m, 2H), 7.02 (d, J = 9.0

Hz, 3H), 6.75 (dd, J = 8.9, 3.1 Hz, 1H), 3.81 (s, 3H), 3.73 (s, 3H).

4-Amino-3-(3-bromophenyl)-N-(2,5-dimethoxyphenyl)-2-thioxo-

2,3-dihydrothiazole-5-carboxamide (19f). 2-Cyano-N-(2,5-

dimethoxyphenyl)acetamide (8i), 1-bromo-3-isothiocyanatobenzene

(18f), and sulfur (12) were reacted using a procedure similar to the

synthesis of 19a, aﬀording compound 19f as a yellow solid (48%

yield).¹H NMR (600 MHz, DMSO-d₆) δ 8.43 (s, 1H), 7.78 (ddd, J =

8.1, 2.0, 1.0 Hz, 1H), 7.72 (t, J = 2.0 Hz, 1H), 7.57 (t, J = 8.0 Hz,

1H), 7.43 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 7.30 (d, J = 3.1 Hz, 1H),

7.02 (s, 2H), 6.98 (d, J = 9.0 Hz, 1H), 6.72 (dd, J = 8.9, 3.1 Hz, 1H),

3.78 (s, 3H), 3.70 (s, 3H).

General Procedure for the Preparation of 20a−20f. 4-(2-

Chloroacetamido)-3-(3-cyanophenyl)-N-(2,5-dimethoxyphenyl)-2-

thioxo-2,3-dihydrothiazole-5-carboxamide (20a). To a solution of

4-amino-3-(3-cyanophenyl)-N-(2,5-dimethoxyphenyl)-2-thioxo-2,3-

dihydrothiazole-5-carboxamide (19a, 0.10 g, 0.24 mmol, 1.0 equiv) in

CH₃CN (6 mL) was added 2-chloroacetyl chloride (0.13 g, 1.21

mmol, 5.0 equiv) in the microwave tube. The resulting mixture was

stirred at 65 °C for 1 h. Upon completion of the reaction, the reaction

solution concentrated by a rotary evaporator gave the crude conduct,

which was puriﬁed by silica gel chromatography (DCM/methanol,

80:1) to aﬀord 20a (0.02 g, yield 17%) as a light-yellow solid. mp

1

180−182 °C. H NMR (400 MHz, DMSO-d₆) δ 11.02 (s, 1H), 8.98

(s, 1H), 8.05 (dt, J = 7.9, 1.3 Hz, 1H), 7.90 (d, J = 1.9 Hz, 1H), 7.83−

7.74 (m, 2H), 7.71−7.65 (m, 1H), 7.02 (d, J = 8.9 Hz, 1H), 6.71 (dd,

J = 8.9, 3.1 Hz, 1H), 4.06 (s, 2H), 3.80 (s, 3H), 3.71 (s, 3H). ¹³C

NMR (126 MHz, DMSO-d₆) δ 186.5, 167.2, 155.4, 153.0, 142.8,

136.0, 134.0, 133.9, 132.5, 130.9, 126.6, 117.7, 112.4, 111.8, 109.0,

107.0, 56.4, 55.4, 41.5. HRMS (AP-ESI) m/z calcd for

C₂₁H₁₈ClN₄O₄S₂[M + H]⁺, 489.0453; found, 489.0454. Retention

time: 3.6 min, eluted with 70% methanol/30% water.

4-(2-Chloroacetamido)-N-(2,5-dimethoxyphenyl)-2-thioxo-3-(3-

(triﬂuoromethyl)phenyl)-2,3-dihydrothiazole-5-carboxamide

(20b). 4-Amino-N-(2,5-dimethoxyphenyl)-2-thioxo-3-(3-

(triﬂuoromethyl)phenyl)-2,3-dihydrothiazole-5-carboxamide (19b)

and 2-chloroacetyl chloride (commercially available) were reacted

using a procedure similar to the synthesis of 20a, aﬀording compound

Hz, 2H), 7.85−7.73 (m, 2H), 7.27 (d, J = 3.1 Hz, 1H), 7.07 (s, 2H),

6.98 (d, J = 9.0 Hz, 1H), 6.71 (dd, J = 9.0, 3.1 Hz, 1H), 3.77 (s, 3H),

3.69 (s, 3H).

4-Amino-N-(2,5-dimethoxyphenyl)-2-thioxo-3-(3-

(triﬂuoromethyl)phenyl)-2,3-dihydrothiazole-5-carboxamide

(19b). 2-Cyano-N-(2,5-dimethoxyphenyl)acetamide (8i), 1-isothio-

cyanato-3-(triﬂuoromethyl)benzene (18b), and sulfur (12) were

1

20b as a light-yellow solid (27% yield). mp 183−185 °C. H NMR

(600 MHz, DMSO-d₆) δ 11.04 (s, 1H), 8.97 (s, 1H), 7.94−7.90 (m,

1H), 7.80 (t, J = 8.0 Hz, 1H), 7.77−7.71 (m, 2H), 7.62 (d, J = 8.2 Hz,

1H), 7.01 (d, J = 9.0 Hz, 1H), 6.69 (dd, J = 8.9, 3.1 Hz, 1H), 4.05−

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3.95 (m, 2H), 3.78 (s, 3H), 3.69 (s, 3H). ¹³C NMR (126 MHz,

DMSO-d₆) δ 186.6, 167.2, 155.4, 153.0, 142.8, 135.9, 133.5, 133.1,

130.8, 130.5, 130.2, 129.9, 126.9, 126.6, 125.6, 124.6, 122.4, 118.7,

111.8, 109.0, 107.0, 56.4, 55.4, 41.4. HRMS (AP-ESI) m/z calcd for

C₂₁H₁₈ClF₃N₃O₄S₂[M + H]⁺, 532.0374; found, 532.0370. Retention

time: 6.8 min, eluted with 70% methanol/30% water.

treated by adding hydrogen atoms, deleting water molecules, and

generating het states using Epik and an optimization and

minimization process using Maestro/Protein Preparation Wizard.

The molecular structure was generated with the Maestro/LigPrep and

other docking parameters were kept to the default values. Molecular

docking was carried out via the Maestro/Ligand-docking module and

Covalent docking module.

MALT1 Expression and Puriﬁcation. MALT1 (340-789) was

fused with a leucine zipper sequence from GCN4 (251-281) in the N-

terminus (LZ-MALT1). N-terminal his-tagged LZ-MALT1 was

expressed in Escherichia coli and puriﬁed by Ni-NTA aﬃnity

chromatography (Qiagen, Valencia, CA) followed by gel ﬁltration

chromatography with Superdex 200 HR 10/300 (GE Healthcare,

U.K.) in a buﬀer containing 20 mM Tris (pH 7.5), 150 mM NaCl,

and 5 mM DTT.

High-Throughput Screening for MALT1 Proteolytic Activity

Inhibitors. The screening consisted of a 20 μL reaction in 384-well

black plates (Greiner Bio One, Wemmel, Belgium, catalogue

#784076) with 120 nM LZ-MALT1, 20 μM Ac-LVSR-AMC, and

12.5 μM test compound in buﬀer A (20 mM HEPES (pH 7.5), 10

mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM DTT, and 0.01%

Triton X-100). The reactions were measured with excitation/emission

wavelengths of 360/465 nm using an Envision Multilabel Reader

(PerkinElmer, Waltham, MA). The positive hits were validated in

concentration−response experiments to determine IC₅₀of the

compounds.

Cell Culture. The GCB-DLBCL cell line OCI-LY1 was grown in

Iscove’s medium supplemented with 20% FBS and penicillin G/

streptomycin. DLBCL cell lines HBL-1 and TMD8 were cultured in

RPMI medium with 10% FBS, 2 mM glutamine, 10 mM HEPES, and

penicillin G/streptomycin. All cell lines were cultured at 37 °C in a

humidiﬁed atmosphere of 5% CO₂. Cell lines were authenticated by

single-nucleotide polymorphism proﬁling.

Growth-Inhibition Determination. Exponentially growing

DLBCL cells (1 × 10⁴) in 100 μL of medium were seeded into

black 96-well microtiter plates (Corning, cat. no. 3603) and incubated

in medium containing either vehicle control (DMSO) or MALT1

inhibitors (in 2-fold serial dilutions starting at 100 μM) for 72 h at 37

°C in three replicates on the same plate. Then, 100 μL of CellTiter-

Glo reagent was added to each well. The luminescence signal in each

well was measured 1 h later in a plate reader (SpectraMax Paradigm,

Molecular Devices). The percentage of cell growth inhibition was

calculated by comparing luminescence readings obtained from treated

and control cells, accounting for the initial cell population. GraphPad

Prism software was used to determine GI₅₀values.

Flow Cytometry. DLBCL cells were treated with compounds at

the indicated concentrations. After 24 h, cells were collected, washed

with PBS, resuspended in binding buﬀer, and stained with Annexin V-

FITC (KeyGen Biotech, KGA703) and propidium iodide (KeyGen

Biotech, KGA703) at 1:100 dilution for 15 min at room temperature

in the dark. Signals were collected by a MACSQuant analyzer and

data were analyzed using FlowJo (Ashland, OR). The percentage of

apoptotic cells was the sum of early (Annexin V⁺/PI⁻) and late

apoptotic cells (Annexin V⁺/PI⁺).

Western Blotting. Equal amounts of total protein (20−75 μg)

were separated on sodium dodecyl sulfate−polyacrylamide gel

electrophoresis (SDS-PAGE) and electrotransferred onto PVDF

membranes. Membranes were incubated with primary antibodies

(RelB, IκBα, and phosphorylated IκBα from Cell Signaling Tech,

Danvers, MA), followed by secondary antibodies conjugated to

horseradish peroxidase, which were detected by chemiluminescence

(Pierce, Thermo Scientiﬁc, Rockford, IL).

Pharmacokinetics. All experiments involving laboratory animals

were performed with the approval of the Animal Institute Committee

of Changhai Hospital, the Second Military Medical University, and

the SIMM Laboratory Animal Center ethics committee. Compound

20c (5% DMSO + 5% Tween-80 in 90% saline) was subjected to PK

studies in ICR (CD-1) mice. Compound 20c was administered via

the oral route at 10 mg/kg, ip route at 1 mg/kg, and iv route at 1 mg/

kg in CD-1 mice. After oral, intraperitoneal, and intravenous

4-(2-Chloroacetamido)-N-(2,5-dimethoxyphenyl)-2-thioxo-3-(4-

(triﬂuoromethyl)phenyl)-2,3-dihydrothiazole-5-carboxamide (20c).

4-Amino-N-(2,5-dimethoxyphenyl)-2-thioxo-3-(4-(triﬂuoromethyl)-

phenyl)-2,3-dihydrothiazole-5-carboxamide (19c) and 2-chloroacetyl

chloride (commercially available) were reacted using a procedure

similar to the synthesis of 20a, aﬀording compound 20c as a light-

1

yellow solid (30% yield). mp 171−173 °C. H NMR (600 MHz,

DMSO-d₆) δ 10.97 (s, 1H), 8.99 (s, 1H), 7.97 (d, J = 8.2 Hz, 2H),

7.79−7.75 (m, 1H), 7.57 (d, J = 8.2 Hz, 2H), 7.02 (d, J = 9.0 Hz,

1H), 6.71 (dd, J = 8.9, 3.1 Hz, 1H), 4.05 (s, 2H), 3.80 (s, 3H), 3.71

(s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 186.3, 167.2, 155.4,

153.0, 142.8, 138.8, 133.4, 130.3, 130.1, 129.8, 126.6, 124.8, 122.6,

118.8, 111.8, 109.0, 107.0, 56.4, 55.4, 41.5. HRMS (AP-ESI) m/z

calcd for C₂₁H₁₈ClF₃N₃O₄S₂[M + H]⁺, 532.0374; found, 532.0370.

Retention time: 7.3 min, eluted with 70% methanol/30% water.

4-(2-Chloroacetamido)-3-(4-chlorophenyl)-N-(2,5-dimethoxy-

phenyl)-2-thioxo-2,3-dihydrothiazole-5-carboxamide (20d). 4-

Amino-3-(4-chlorophenyl)-N-(2,5-dimethoxyphenyl)-2-thioxo-2,3-di-

hydrothiazole-5-carboxamide (19d) and 2-chloroacetyl chloride

(commercially available) were reacted using a procedure similar to

the synthesis of 20a, aﬀording compound 20d as a light-yellow solid

1

(15% yield). mp 154−156 °C. H NMR (600 MHz, DMSO-d₆) δ

11.04 (s, 1H), 8.97 (s, 1H), 8.02−7.89 (m, 1H), 7.80 (t, J = 8.0 Hz,

1H), 7.77−7.71 (m, 2H), 7.62 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 9.0

Hz, 1H), 6.69 (dd, J = 8.9, 3.1 Hz, 1H), 4.07−3.96 (m, 2H), 3.78 (s,

3H), 3.69 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 186.4, 167.2,

155.4, 153.0, 142.7, 134.7, 134.2, 130.4, 129.6, 126.7, 111.8, 108.9,

106.9, 56.4, 55.4, 41.7. HRMS (AP-ESI) m/z calcd for

C₂₀H₁₈Cl₂N₃O₄S₂[M + H]⁺, 498.0110; found, 498.0103. Retention

time: 6.3 min, eluted with 70% methanol/30% water.

4-(2-Chloroacetamido)-N-(2,5-dimethoxyphenyl)-3-(3-ﬂuoro-

phenyl)-2-thioxo-2,3-dihydrothiazole-5-carboxamide (20e). 4-

Amino-N-(2,5-dimethoxyphenyl)-3-(3-ﬂuorophenyl)-2-thioxo-2,3-di-

hydrothiazole-5-carboxamide (19e) and 2-chloroacetyl chloride

(commercially available) were reacted using a procedure similar to

the synthesis of 20a, aﬀording compound 20e as a light-yellow solid

(33% yield). mp 178−180 °C. ¹H NMR (600 MHz, DMSO-d₆) δ (s,

1H), 8.97 (s, 1H), 7.76 (d, J = 3.1 Hz, 1H), 7.61 (td, J = 8.2, 6.3 Hz,

1H), 7.42 (tdd, J = 8.6, 2.6, 0.9 Hz, 1H), 7.25 (dt, J = 9.4, 2.3 Hz,

1H), 7.16−7.11 (m, 1H), 7.01 (d, J = 9.0 Hz, 1H), 6.69 (dd, J = 8.9,

3.1 Hz, 1H), 4.05 (s, 2H), 3.79 (s, 3H), 3.69 (s, 3H).¹³C NMR (126

MHz, DMSO-d₆) δ 186.8, 167.7, 153.5, 143.2, 131.6, 129.1, 125.3,

116.8, 116.6, 112.2, 109.4, 107.3, 56.9, 55.9, 42.0. HRMS (AP-ESI)

m/z calcd for C₂₀H₁₆ClFN₃O₄S₂[M − H]⁻, 480.0260; found,

480.0253. Retention time: 4.5 min, eluted with 70% methanol/30%

water.

3-(3-Bromophenyl)-4-(2-chloroacetamido)-N-(2,5-dimethoxy-

phenyl)-2-thioxo-2,3-dihydrothiazole-5-carboxamide (20f). 4-

Amino-3-(3-bromophenyl)-N-(2,5-dimethoxyphenyl)-2-thioxo-2,3-di-

hydrothiazole-5-carboxamide (19f) and 2-chloroacetyl chloride

(commercially available) were reacted using a procedure similar to

the synthesis of 20a, aﬀording compound 20f as a light-yellow solid

1

(18% yield). mp 178−180 °C. H NMR (600 MHz, DMSO-d₆) δ

10.98 (s, 1H), 8.97 (s, 1H), 7.77 (d, J = 7.1 Hz, 2H), 7.61−7.50 (m,

2H), 7.31 (d, J = 8.0 Hz, 1H), 7.02 (d, J = 8.9 Hz, 1H), 6.70 (dd, J =

9.0, 3.1 Hz, 1H), 4.06 (s, 2H), 3.80 (s, 3H), 3.71 (s, 3H). ¹³C NMR

(126 MHz, DMSO-d₆) δ 186.9, 167.7, 153.5, 143.2, 137.0, 133.5,

131.8, 128.3, 127.2, 122.0, 112.3, 109.4, 107.3, 56.9, 55.9, 42.0.

HRMS (AP-ESI) m/z calcd for C₂₀H₁₈BrClN₃O₄S₂[M + H]⁺,

541.9605; found, 541.9614. Retention time: 6.3 min, eluted with 70%

methanol/30% water.

Molecular Docking Study. The crystal structure of MALT1

(PDB codes: 4I1R, 3V4O, and 3UO8) was obtained from the Protein

Data Bank. Before the docking process, the structure of protein was

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administration, blood samples were collected from each animal via

jugular vein and stored in ice (0−4 °C) at the speciﬁc time points.

Plasma was separated from the blood by centrifugation and stored in a

freezer at −80 °C. All samples for the tested compounds were

analyzed by LC−MS/MS.

China; CAS Key Laboratory of Tissue Microenvironment and

Tumor, Shanghai Institute of Nutrition and Health, Chinese

Academy of Sciences, Shanghai 200031, China

Chunpu Li − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China; University of Chinese

Academy of Sciences, Beijing 100049, China

Haolan Yu − Department of Urology, Changhai Hospital,

Second Military Medical University, Shanghai 200043,

China; CAS Key Laboratory of Tissue Microenvironment and

Tumor, Shanghai Institute of Nutrition and Health, Chinese

Academy of Sciences, Shanghai 200031, China

Xiaohui Wei − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China; University of Chinese

Academy of Sciences, Beijing 100049, China

Xuyi Liu − State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, China; University of Chinese Academy of

Sciences, Beijing 100049, China

Wei Bao − Department of Urology, Changhai Hospital, Second

Military Medical University, Shanghai 200043, China; CAS

Key Laboratory of Tissue Microenvironment and Tumor,

Shanghai Institute of Nutrition and Health, Chinese

Academy of Sciences, Shanghai 200031, China

In Vivo Antitumor Activity Assay in the TMD8 Xenograft

Model. TMD8 cells (5 × 10⁶) were inoculated subcutaneously in the

right ﬂank of 6 week-old female NOD^{prkdc−/−IL‑2Rg−/−}(NCG) mice

(GemPharmatech Co., Ltd). Ten days after injection, tumors were

palpable and mice were randomized into treatment and control

groups (eight mice per group). The treatment groups received

speciﬁed concentrations of compounds by intraperitoneal admin-

istration, and the blank control group received intraperitoneal

administration of an equal volume of HPBCD (20% PEG 400).

During treatment, subcutaneous tumors were measured with a digital

caliper every 2 days, and body weight was monitored regularly. Tumor

volume (V) was estimated using the equation (V = ab²/2, where a

and b stand for the longest and shortest diameters, respectively).

After treatment, mice were sacriﬁced and dissected to weigh the

tumor tissues and to examine the internal organ injury by macroscopic

analysis. TGI was calculated according to the following formula: TGI

= (the mean tumor weight of the control group − the mean tumor

weight of the treated group)/the mean tumor weight of the control

group. All experiments were performed according to the institutional

ethical guidelines on animal care and approved by the Institute

Animal Care and Use Committee at Shanghai Institute of Materia

Medica.

Yuqiang Shi − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China; University of Chinese

Academy of Sciences, Beijing 100049, China

Xiaochen Sun − Department of Urology, Changhai Hospital,

Second Military Medical University, Shanghai 200043,

China; CAS Key Laboratory of Tissue Microenvironment and

Tumor, Shanghai Institute of Nutrition and Health, Chinese

Academy of Sciences, Shanghai 200031, China

Mirzadavlat Khamrakulov − State Key Laboratory of Drug

Research, Shanghai Institute of Materia Medica, Chinese

Academy of Sciences, Shanghai 201203, China; University of

Chinese Academy of Sciences, Beijing 100049, China

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00466.

■

sı

Chemical structures of compounds F1−F36; predicted

binding mode of compound F31 in the paracaspase

domain of MALT1; tumor cell apoptosis-inducing

activity of compounds 15c and 15e; H NMR and ¹³C

1

NMR spectra of all target compounds; HPLC and

HRMS spectra of representative compounds (PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00466

■

Chenghua Yang − Department of Urology, Changhai

Hospital, Second Military Medical University, Shanghai

200043, China; CAS Key Laboratory of Tissue

Microenvironment and Tumor, Shanghai Institute of

Nutrition and Health, Chinese Academy of Sciences,

Shanghai 200031, China; Phone: +86 21 31162795;

Email: chenghua-yang@qq.com

Hong Liu − State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, China; University of Chinese Academy of

Sciences, Beijing 100049, China; School of Pharmaceutical

Science and Technology, Hangzhou Institute for Advanced

Study, University of Chinese Academy of Sciences, Hangzhou

310024, China; orcid.org/0000-0003-3685-6268;

Phone: +86 21 50807042; Email: hliu@mail.shcnc.ac.cn

Author Contributions

^∥X.L., C.S., C.L., and H.Y. contributed equally to this work.

The manuscript was written by X.L. Synthesis of compounds

was performed by X.L. and X.W. High-throughput screening

assay was developed by C.S., and biological function testing of

all the MALT1 inhibitors was performed by C.S., H.Y., and

W.B. Docking analysis was performed by C.L. C.Y. and H.L.

supervised the project. All authors have given approval to the

ﬁnal version of the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by the National Natural Science

Foundation of China (grant nos. 21907102, 21632008,

81620108027, 21877118, 91953108, 21977106, and

81903434) and Science and Technology Commission of

Shanghai Municipality (18JC1411304, 18431907100,

19431908100, and 19431901000). This work was also

supported by the National Natural Science Foundation of

China (grant nos. 81473245 and 81772695), “100 Talent

Authors

Xuewu Liang − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China; University of Chinese

Academy of Sciences, Beijing 100049, China

Chenxia Sun − Department of Urology, Changhai Hospital,

Second Military Medical University, Shanghai 200043,

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J. Med. Chem. 2021, 64, 9217−9237

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(14) Hahn, M.; Bu

̈

rckert, J.-P.; Luttenberger, C. A.; Klebow, S.;

Program” from Chinese Academy of Science, and the Strategic

Priority Research Program of the Chinese Academy of

Sciences (XDA12020352) to C.Y.

Hess, M.; Al-Maarri, M.; Vogt, M.; Reißig, S.; Hallek, M.; Wienecke-

Baldacchino, A.; Buch, T.; Muller, C. P.; Pallasch, C. P.; Wunderlich,

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suppressor CYLD promotes the development of chronic lymphocytic

leukemia via sustained NF-κ B signaling. Leukemia. 2018, 32, 72−82.

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Rosenwald, A.; Boldrick, J. C.; Sabet, H.; Tran, T.; Yu, X.; Powell, J.

I.; Yang, L.; Marti, G. E.; Moore, T.; Hudson, J., Jr.; Luo, L.; Lewis, D.

B.; Tibshirani, T. R.; Sherlock, G.; Chan, W. C.; Greiner, T. C.;

Weisenburger, D. D.; Armitage, J. O.; Wamke, R.; Levy, R.; Wilson,

W.; Grever, M. R.; Byrd, J. C.; Botstein, D.; Brown, P. O.; Staudt, L.

M. Distinct types of diffuse large B-cell lymphoma identified by gene

expression profiling. Nature 2000, 403, 503−511.

ABBREVIATIONS USED

■

ABC-DLBCL, activated B cell-like diﬀuse large B cell

lymphoma; API2, cellular inhibitor of apoptosis 2; BCL10, B

cell lymphoma 10 protein; CLL, chronic lymphocytic

leukemia; DD, death domain; GCB-DLBCL, germinal center

B cell-like diﬀuse large B cell lymphoma; IKK, IκB kinase;

MALT1, mucosa-associated lymphoid tissue lymphoma trans-

location protein 1

(16) Wilson, W. H.; Young, R. M.; Schmitz, R.; Yang, Y.; Pittaluga,

S.; Wright, G.; Lih, C.-J.; Williams, P. M.; Shaffer, A. L.; Gerecitano,

J.; de Vos, S.; Goy, A.; Kenkre, V. P.; Barr, P. M.; Blum, K. A.;

Shustov, A.; Advani, R.; Fowler, N. H.; Vose, J. M.; Elstrom, R. L.;

Habermann, T. M.; Barrientos, J. C.; McGreivy, J.; Fardis, M.; Chang,

B. Y.; Clow, F.; Munneke, B.; Moussa, D.; Beaupre, D. M.; Staudt, L.

M. Targeting B cell receptor signaling with ibrutinib in diffuse large B

cell lymphoma. Nat. Med. 2015, 21, 922−926.

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Article Doi

Identification of Novel Fused Heteroaromatics-Based MALT1 Inhibitors by High-Throughput Screening to Treat B Cell Lymphoma

DOI: 10.1021/acs.jmedchem.1c00466

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