Discovery of Novel Transient Receptor Potential Vanilloid 4 (TRPV4) Agonists as Regulators of Chondrogenic Differentiation: Identification of Quinazolin-4(3 H)-ones and in Vivo Studies on a Surgically Induced Rat Model of Osteoarthritis

Article

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Cite This: J. Med. Chem. 2019, 62, 1468 −1483

Discovery of Novel Transient Receptor Potential Vanilloid 4 (TRPV4)

Agonists as Regulators of Chondrogenic Diﬀerentiation:

Identiﬁcation of Quinazolin-4(3H)‑ones and in Vivo Studies on a

Surgically Induced Rat Model of Osteoarthritis

Masakazu Atobe,*^,†,∇Takamichi Nagami,^‡,∇Shuji Muramatsu,^‡,⊥Takeshi Ohno,^‡,⊥

Masayuki Kitagawa,^‡,⊥Hiroko Suzuki,^§Masashi Ishiguro,^§Atsushi Watanabe,^∥and Masashi Kawanishi^†

‡

^†Laboratory for Medicinal Chemistry, Pharmaceutical Research Center, Laboratory for Pharmacology, Pharmaceutical Research

Center, and ^§Laboratory for Safety Assessment & ADME, Pharmaceutical Research Center, Asahi Kasei Pharma Corporation, 632-1

Mifuku, Izunokuni, Shizuoka 410-2321, Japan

^∥Medical Technology & Material Laboratory, Medical Products Development Division, Asahi Kasei Medical Corporation, 632-1

Mifuku, Izunokuni, Shizuoka 410-2321, Japan

S

* Supporting Information

ABSTRACT: Osteoarthritis (OA) is a degenerative disease

characterized by joint destruction and loss of cartilage. There

are many unmet needs in the treatment of OA and there are

few promising candidates for disease-modifying OA drugs,

particularly, anabolic agents. Here, we describe the identi-

ﬁcation of novel quinazolin-4(3H)-one derivatives, which

stimulate chondrocyte cartilage matrix production via TRPV4 and mitigate damaged articular cartilage. We successfully

identiﬁed the water-soluble, highly potent quinazolin-4(3H)-one derivative 36 and studied its intra-articular physicochemical

proﬁle to use in in vivo surgical OA model studies. Compound 36·HCl provided relief from OA damage in a rat medial meniscal

tear (MT) model. Speciﬁcally, 36·HCl dose-dependently suppressed cartilage degradation and enhanced the messenger RNA

expression of aggrecan and SOX9 in cartilage isolated from MT-operated rat knees compared with knees treated with vehicle.

These results suggest that 36 induces anabolic changes in articular cartilage and consequently reduces OA progression.

and enzymes⁶or on anabolic factors that may stimulate the

INTRODUCTION

■

repair of cartilage lesions.⁷The targeting of anabolic factors

Osteoarthritis (OA) is a degenerative disease that typically

aﬀects the elderly and is the most common form of arthritis. OA

represents an attractive therapeutic approach for OA. In

is characterized by joint destruction and loss of cartilage,¹the

addition, a number of growth factors have been reported to

have anabolic eﬀects on cartilage; for example, FGF18 has

slippery tissue that covers the ends of bones in a joint. Healthy

shown eﬃcacy in preclinical models of traumatic cartilage

cartilage allows bones to glide over one another and absorbs

injury.⁸To our knowledge, however, there are very few reports⁹

energy from the shock of physical movement.²In OA, the

of the eﬃcacy of small molecules in animal models in which

cartilage damage is due to joint instability. Such models are

representative of joint failure in OA.

The SRY (sex-related Y)-type high-mobility group box

surface layer of cartilage breaks down and wears away, allowing

bones under the cartilage to rub together, causing pain, swelling,

and loss of motion of the joint. In addition, the joint may lose its

normal shape and bone spurs, called osteophytes, may grow on

“SOX9” is a transcription factor that is essential for chondrocyte

the edges of the joint. Fragments of bone or cartilage can break

diﬀerentiation and chondrocyte-speciﬁc gene expression of

oﬀ and ﬂoat inside the joint space, causing more pain and

molecules,^10,11such as collagen type II,¹²IX,¹³and XI¹⁴and

aggrecan.¹⁵Sox9 is involved in the expression of SOX5 and

SOX6, both of which form a transcriptional complex with SOX9

and control the expression of type II collagen and aggrecan.¹⁶

However, the precise mechanism of SOX9 activation during

chondrogenesis is not fully understood. In our previous study,

we performed functional gene screening to identify genes that

activate SOX9-dependent transcription, using full-length

damage.^1,2Various treatments and therapies are available for

OA.³The existing medications, which are nonsteroidal anti-

inﬂammatory drugs and corticosteroids, are analgesics that help

to relieve pain and act on the immune system by blocking the

production of compounds that trigger allergic and inﬂammatory

reactions. Some OA treatments also target processes believed to

cause the disease.⁴Severe cases of OA are treated with

arthroplasty, which involves reconstruction or replacement of

the diseased joint and realignment of the joint.⁵

Recent eﬀorts to identify agents that alter the progression of

OA have focused on inhibiting the catabolic cascade of cytokines

Received: October 18, 2018

Published: January 10, 2019

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Figure 1. GSK compounds 1 and 2 and 4αPDD 3.

complementary DNA (cDNA) libraries generated from a

murine chondrogenic cell line, ATDC5.¹⁷In that study, we

identiﬁed the protein TRPV4 through its eﬀect on SOX9-

dependent transcription. Our data suggested that TRPV4 plays

an important role in early chondrogenesis. Thus, examining the

role of TRPV4 during hypertrophic diﬀerentiation in late

chondrogenesis would be informative because SOX9 functions

as a negative regulator in this process.

RESULTS AND DISCUSSION

Optimization of Quinazolin-4(3H)-one 4 for Use in in

Vivo Studies. High-throughput screening using SOX9 reporter

assays with ATDC5 cells and our 350 000 inhouse compound

library identiﬁed 24 hit compounds (0.007% hit rate).

Quinazolin-4(3H)-one 4 showed moderate SOX9 reporter

activity (Figure 2). The potency of 4 was canceled with

■

TRPV4 is widely expressed in epithelial cells and is found in

the brain, endothelium, liver, dorsal root ganglia, bladder, skin,

heart, and cartilage.¹⁸The literature reports the contribution of

TRPV4 in several pathological conditions, such as hypotonic

hyperalgesia, thermal hyperalgesia, asthma, and neuropathic

pain.¹⁹Several small-molecule inhibitors of TRPV4 have been

identiﬁed.²⁰Notably, researchers at Glaxo Smith Klein (GSK)

showed that the piperazine-linked TRPV4 agonist

GSK1016790A (1) exhibits higher potency and selectivity

against TRPV1 than for 4αPDD (3), a semisynthetic nontumor

promoter phorbol ester (Figure 1).²¹Compound (2) with a

similar structure but lacking a piperazine ring has also been

described.²²GSK researchers disclosed that the intravenous

administration of 1 (Figure 1) led to a dose-dependent

reduction in blood pressure, circulatory collapse, and death

and concluded that TRPV4 is toxic to humans due to its

agonistic modulation of many physiological functions.²³

Figure 2. HTS hit compound 4.

ruthenium red, which is TRPV4 antagonist; thus, SOX9 reporter

activity derives from TRPV4 agonistic activity. TRPV4 conducts

Ca²⁺and Na⁺across the plasma membrane of various cell types

with a 6:1 selectivity under physiological conditions,²⁴which

normally enhances cellular excitability. Consequently, com-

pound 4 was tested for activity using the TRPV4 Ca²⁺assay

25

(EC_max19% at 10 μM). Several sulfonamide-type hit

Given these advantages and disadvantages of TRPV4 agonists,

our aim is to identify a highly potent TRPV4 agonistic

modulator with good physicochemical properties for intra-

articular administration into exposed joint cartilages. We intend

to avoid oral and intravenous administration routes so that the

entire body is not exposed to the TRPV4 agonistic modulator.

We ﬁrst conﬁrmed whether GSK-patented compound 1 or

phorbol ester 3 are appropriate therapeutic compounds. Our

results suggested that the high lipophilicity and large molecular

weights of 1 and 3 make them unsuitable for use in in vivo

validation studies. Here, we describe the optimization of our

novel TRPV4 agonist and its in vivo evaluation.

compounds showed moderate SOX9 reporter activity, but all

compounds showed relatively high lipophilic proﬁles and had

structures similar to 1 and 2 (structures not shown). We

therefore chose compound 4 for further optimization to improve

its TRPV4 agonistic activity and solubility in aqueous buﬀer to

work toward an injectable compound.

Structure−activity relationship (SAR) studies of 4 were

carried out to obtain more potent compounds (Table 1). We

ﬁrst conducted SAR studies on the phenyl ring. Compound 5

(R⁴= Me) showed moderate activity whereas 6 (R⁵= Me) and 7

(R⁶= Me) showed no potency, thereby directing us to R⁴as a

potential position for other substituents. Compound 8 (R⁴=

CO₂Et) showed activity similar to that of 5. The replacement of

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Table 1. Initial Optimization of HTS Hit Compound 4

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Table 1. continued

Cl by F at the R²position improved activity and gave an EC₅₀of

920 nM in the Ca²⁺assay. We next studied the SAR of the R²

position and observed decreased activity for compounds 10 (R²

= H), 11 (R²= Br), and 12 (R²= OMe). Replacement of the

piperazine unit to give compounds 13, 14, and 15 also resulted

in decreased potency. Further exploration of potential

substituents on the piperazine unit in compounds 16−25 also

resulted in reduced or lost activity. In contrast, the optimization

of R⁴in compounds 26, 27, and 28 resulted in good potency

(2500, 2500, and 625 nM, respectively) and 29 showed excellent

activity at 156 nM.

Compound 32 (R³= F) showed activity similar to that observed

using the SOX9 reporter assay (625 nM), and its activity in the

hTRPV4 Ca²⁺assay was slightly lower than that of 28. Thus, R⁴

is the best position for a ﬂuorine atom.

We attempted further optimization using rapid solid-phase

synthesis by replacing the CF₃with other substituted groups.

The results are summarized in Table 3. Compound 33, bearing a

tetrahydropyran group, showed slightly less activity than 34 but

had good solubility and a lower protein binding ratio than that of

34. Compound 35 exhibited signiﬁcantly increased potency

(EC_max: 39 nM in the SOX9 reporter assay). Compound 36,

bearing a 4-phenyl group, showed the best SOX9 reporter assay

activity and hTRPV4 Ca²⁺assay results. Compound 36 was

inactive against TRPV1 channels, the TRP superfamily member

closest to TRPV4 on the basis of sequence homology. We tested

36 using the Ames test and ATP assay using HL-60 cell viability

because these cells provide a sensitive assay for evaluating the

cytotoxicities of compounds.²⁷No toxicity was observed. Also,

36 exhibited good solubility in saline solution and a moderate

protein binding ratio. Therefore, 36 was chosen as the most

promising compound for testing in an animal OA model.

Evaluation of Compounds Using the Human Articular

Chondrocyte (NHAC) Cell Assay. Next, we screened selected

compounds for in vivo study by evaluating their cell activities

using the normal human articular chondrocyte (NHAC) cell

assay.²⁸Chondrocytes are specialized cells that produce and

maintain the extracellular matrix of cartilage and are the only

cellular component of cartilage. The results are shown in Figure

3. Compound 2 was not active below 500 nM, whereas 29 and

36 showed suﬃcient activity to generate chondrocyte

extracellular matrix stained by Alcian blue, suggesting their

possible chondrogenesis activity. It is noteworthy that 36

We next investigated potentially important positions on the

quinazolin-4(3H)-one core of 28 using a ﬂuorine scanning

strategy²⁶(Table 2). Compounds 30 (R¹= F) and 31 (R²= F)

showed moderate activity (1250 and 2500 nM, respectively).

Table 2. Results of the Fluorine Scanning Strategy

hTRPV1

SOX9

hTRPV4

Ca²⁺

(10 μM)

(%)

reporter assay Ca²⁺EC₅₀

cpd. R¹

R²

R³

R⁴

EC_max(nM)

(nM)

28

30

31

32

-H

-F

-H

-F

-H

-F

625

1250

2500

625

280

1180

2060

663

2.2

3.7

5.0

6.2

-H

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Table 3. Final Optimization Results

concentration of each test compound in the lavage from the

articular joint was measured by liquid chromatography with

tandem mass spectrometry and then converted to the

concentration in the joint using the ratio of the total protein

concentration in the lavage. The analytical quantitation limits of

1, 29·HCl, and 36·HCl were 0.3, 1, and 1 nmol/L, respectively,

as shown in Figure 4. GSK compound 1 showed long-term PK

Figure 3. Human chondrocyte assay results following 30 min

stimulation with TRPV4 agonists.

showed activity at 62.5 nM, as determined by the Alcian blue

intensity, which is the highest activity and thus the highest

potency of the tested compounds.

Measurement of the Concentrations of 1, 29·HCl, and

36·HCl in Articular Cartilage. On the basis of our concept of

an ideal TRPV4 agonist for treating OA, the three drug

candidates were injected into articular joints directly (SI Figure

1). Injected TRPV4 agonist quickly stimulates ion channels to

activate chondrocytes to produce extracellular matrix. The

matrix is transferred to the systemic blood and quickly

eliminated from the body. This “soft-drug” concept requires

short-term action and not long residency in the articular

cartilage, as indicated by our cell studies. We therefore prepared

29·HCl and 36·HCl for the injection and monitored the

pharmacokinetics (PK) of 1, 29·HCl, and 36·HCl in joints by

injecting the compounds into the knee joints of normal rats.

After euthanizing the rats, the joints were washed by injecting

saline and the synovial ﬂuid lavage (SFL) was collected. The

Figure 4. Concentration study in synovial ﬂuid.

due to its high lipophilicity. The residence time of a drug in the

joint was derived from protein binding against albumin.²⁹There

is an equilibrium between serum and joint cartilage due to

passive diﬀusion. Unbound drugs can permeate the cell

membrane or cartilage and should have low lipophilicities, and

thus GSK compound 1 does not meet our criteria (Figure 4). We

then performed the same assay with 29·HCl and 36·HCl, and

the results are shown Figure 4. Compound 36·HCl provided an

ideal PK plot, quickly leaving the joint cavity (within 30 min),

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and thus 36·HCl exhibited the most promising proﬁle for

surgical OA model studies.

Induction of OA in an MT Model and Drug Treatment.

Osteoarthritis models have classically been categorized into

spontaneous and induced models.³⁰⁻³⁶For simplicity, the

models have been grouped into two basic classes of OA: primary

osteoarthritis (POA) and post-traumatic osteoarthritis (PTOA,

a subcategory of secondary OA). A large number of surgically

induced OA models have been reported and include the medial

meniscal tear (MT) model.³⁷There is a strong association

between meniscal damage and cartilage loss, making the rat MT

model an attractive preclinical model for degenerative joint

diseases.

We evaluated whether our TRPV4 agonist 36·HCl can

facilitate relief from cartilage damage caused by OA by testing

the eﬃcacy of 36·HCl in an in vivo rat MT model. In this model,

a full-thickness cut in the medial meniscus leads to joint

instability and progressive development of OA characterized by

proteoglycan loss, cartilage ﬁbrillation, chondrocyte death,

eventual damage to the subchondral bone, and formation of

osteophytes.^31,38

The results of the rat MT-model studies are shown in Figure 5.

Operated knees intra-articularly received a 50 μL injection of 0,

5, or 10 μM of 36·HCl twice weekly for 3 weeks. Saline was used

as the vehicle. No deaths occurred, and no obvious systemic

eﬀects of 36·HCl treatment were observed by gross visual

inspection of the animals. No signiﬁcant diﬀerences in ﬁnal body

weights were noted among the MT-operated groups of rats.

Histological observation showed that the sham-operated

animals exhibited no cartilage degeneration whereas the vehicle-

treated animals showed signiﬁcant cartilage degeneration. The

anabolic eﬀects of 36·HCl were quantiﬁed by measuring the

length of the damaged area. Collapse of articular cartilage into

the tibial epiphysis was occasionally observed. (Figure 5a). Intra-

articular injection of 36·HCl resulted in a dose-dependent

decrease in the length of the damaged area (Figure 5b,c).

In addition to determining the anabolic eﬀects of 36·HCl at

the articular surface, medial sections were stained with either

Safranin O/Fast Green or AB-PAS and observed microscopi-

cally (Figure 6). Little or no staining was observed following

vehicle administration. In contrast, the administration of 10 μM

36·HCl resulted in an area stained by both Safranin O/Fast

Green and AB-PAS. Histological analysis of the joint sections

indicated that 36·HCl-induced newly generated extracellular

matrix. The anabolic eﬀects observed with 36·HCl are likely due

to its TRPV4 agonistic activity increasing the concentration of

SOX9 in the joint.

Next, we measured aggrecan and SOX9 messenger RNA

(mRNA) expression. A dose-dependent increase in the mRNA

expression of aggrecan (Figure 7a) and SOX9 (Figure 7b) was

observed in the cartilage of rat knee joints injected intra-

articularly with 36·HCl 21 days after MT surgery. These changes

in mRNA expression are likely associated with the decreased

pathological damage of cartilage and suggest that small-molecule

36·HCl induces anabolic changes in articular cartilage and thus

reduces OA progression. A detailed mechanism for these eﬀects

is currently not available but the data are consistent with the in

vivo observations that 36·HCl stimulates an increase in SOX9

and then increases the production of extracellular matrix via

chondrocyte diﬀerentiation.

Figure 5. Width of damaged medial tibial cartilage in a rat MT model,

mean SE, n = 5 (sham), n = 15 (vehicle), n = 14 (5 μM), and n = 15

(10 μM) (p value *: p < 0.05 **: p < 0.01 ***: p < 0.001, by Dunnett’s

test). (a) vehicle. (b) 5 μM. (c) 10 μM. Figure 5 Intra-articular injection

of 36·HCl resulted in reduced cartilage lesion area in a rat MT model.

Rats were subjected to meniscal tear and treatment with either the

vehicle alone or with vehicle containing 36·HCl at diﬀerent

concentrations. The width of the damaged area following treatment

with each concentration of 36·HCl. Safranin O and Fast Green-stained

coronal sections of the medial tibial plateau of rats treated with vehicle,

5 μM (b), and 10 μM (c) of 36·HCl.

models. 36·HCl was administrated twice a week for 3 weeks

starting 1 week after MT surgery and dose-dependently reduced

the erosion of tibial plateau cartilage. Although 36·HCl rapidly

leaves the joint cavity, mRNA expression of aggrecan and the

transcription factor SOX9 was enhanced in knee cartilage

isolated from 36·HCl-treated rat MT models. These results

suggest that 36·HCl may promote chondrocyte diﬀerentiation

and extracellular matrix production, thereby reducing cartilage

degradation in rat OA models.

In this report, we demonstrated that 36·HCl promoted

extracellular matrix production in chondrocytes. Intra-articular

injection of 36·HCl reduced cartilage degradation in rat MT

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Figure 6. Medial sections, Safranin O, and Fast Green staining following treatment with vehicle (a), 10 μM of 36·HCl (b); AB-PAS staining following

treatment with vehicle (c), 10 μM of 36·HCl (d).

We next prepared various substituted derivatives of the

piperazine ring using 49c and 49d as the common intermediates.

Piperazines 51a−f were aminated, followed by deprotection and

N-methylation, with typically good yields (Scheme 3).

CHEMISTRY

■

The quinazolin-4(3H)-one derivatives 4−9, 11−15, 26−29,

and 33−36 were synthesized by solid-phase aza-Wittig-

mediated annulation methods^39,40on an e-DOMINO solid-

phase synthesizer invented by us⁴¹(Scheme 1). Azidation of the

easily available anthranilic acid 37 under Sandmeyer conditions

gave ortho azido benzoic acid 38 in 99% yield. Compound 38

was readily coupled using typical amidation conditions with

dicyclohexylcarbodiimide to aﬀord the corresponding polymer-

bound ortho-azide ester 39. Iminophosphorane was synthesized

by exposing 39 to PPh₃in tetrahydrofuran at room temperature,

followed by isocyanate, to give carbodiimide 41. Finally,

treatment with various piperazine derivatives, followed by

intramolecular cyclization and simultaneous cleavage from the

resin, provided the quinazolin-4(3H)-ones 4−9, 11−15, 26−

29, and 33−36. For the use of in vivo studies, 29 and 36 were

treated with 1 M HCl in MeOH solution to give the

corresponding HCl salts, 29·HCl and 36·HCl.

Compounds 10, 17−19, and 30−32 were synthesized using a

liquid-based synthetic route, as shown in Scheme 2.⁴²Isatoic

anhydrides 45a and 45b were prepared from anthranilic acids

44a and 44b with treatment of triphosgene. Other isatoic

anhydrides 45c−e are commercially available. Treatment of

isatoic anhydrides (45) with aniline 46a and 46b, followed by

cyclization with thiocarbonyldiimidazole, aﬀorded the thio-

carbonyl 4(1H)-quinazolinone 48. Chlorination of 48 with

sulfuryl chloride gave 2-chloro-4(3H)-quinazolinone 49, which

was treated with the amines N-methylpiperazine 43a, and 2-

N,N-dimethylaminoethylamine 43e to give the corresponding

2-position-substituted quinazolin-4(3H)-ones 10, 17−19, and

30−32.

CONCLUSIONS

■

We have identiﬁed novel quinazolin-4(3H)-one derivatives that

exhibit anabolic activity toward chondrogenic diﬀerentiation

and provide relief against articular cartilage damage via TRPV4

agonistic stimulation. Highly potent quinazolin-4(3H)-one 36

exhibited good solubility, a promising physicochemical proﬁle,

and high potency in an NHAC cell assay. Intra-articular

injection of 36·HCl suppressed OA progression in rat MT

models. Furthermore, 36·HCl enhanced the mRNA expression

of aggrecan and SOX9 in tibial cartilage isolated from MT rats.

These ﬁndings suggest that TRPV4 agonist 36·HCl stimulates

chondrocyte anabolic changes and helps suppress OA

progression. Our novel approach of promoting new cartilage

formation by using a TRPV4 agonist holds promise as a

treatment approach for OA. In addition, compound 36·HCl is a

promising candidate of the disease-modifying OA drug.

EXPERIMENTAL SECTION

■

General Chemistry Information. NMR spectra were run at 300

MHz on JEOL Co. and at 400 and 600 MHz on Varian Co. As a solvent,

deuterated chloroform was used, unless speciﬁcally described

otherwise. For measurement of chemical shift, tetramethylsilane

(TMS) was employed as an internal standard. Chemical shifts (δ) are

expressed in ppm. Data for proton spectra are reported as follows:

chemical shift (multiplicity [singlet (s), doublet (d), triplet (t), and

multiplet (m)], coupling constants [Hz], and integration). Carbon

spectra were recorded with complete proton decoupling, and the

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chemical shifts are reported in ppm. As a mass spectrometer, the

quadrupole type mass spectrometer, UPLC/SQD system, manufac-

tured by Waters Company was used, and ionization was carried out on

the basis of an electrospray method (ESI) for the measurement. The

liquid chromatography instrument used was Acquity Ultra Performance

LC system. As a separation column, ACQUITY UPLC BEH (C₁₈2.1 ×

50 mm 1.7 μM, Waters Company) was used. With respect to the

examples and the reference examples in which speciﬁc descriptions are

given for LC condition, the measurements were carried out by using any

one of the above described apparatuses and in accordance with the

following solvent condition. In addition, m/z indicates mass spectrum

data (M + H). Flow rate: 0.6 mL/min. Solvent: solution A = water

including 0.1% (v/v) acetic acid, solution B = acetonitrile including

0.1% (v/v) acetic acid. From 0 to 2 min: linear gradient from [solution

A 95% + solution B 5% (v/v)] to [solution A 10% + solution B 90% (v/

v)]. From 2 to 2.5 min: linear gradient from [solution A 10% + solution

B 90% (v/v)] to [solution A 2% + solution B 98% (v/v)]. From 2.5 to

2.6 min: linear gradient from [solution A 2% + solution B 98% (v/v)] to

[solution A 95% + solution B 5% (v/v)]. From 2.6 to 3.2 min:

maintained at [solution A 95% + solution B 5% (v/v)]. Compound

purity was determined using HPLC and is ≥95% for all test compounds.

Solid-Phase Synthesis of the Quinazolin-3-one Derivatives

(4−9, 11−15, 26−29, and 33−36). Preparation of Polymer-Bound

ortho-Azide Ester. Solution of carboxylic acid 38a−d (2.5 equiv) in

CH₂Cl₂(3 mL), HOBt (3.5 equiv) in CH₂Cl₂(2 mL), DIEA, and DIC

(7.0 equiv) were added to a suspension of Wang resin in CH₂Cl₂

suspension with a 10 mL syringe and then agitated at RT for 48 h with

an IKA shaker. Thereafter, the reaction mixture was washed with each

solvent in the following order, respectively: MeOH (3 mL × 3), CH₂Cl₂

(3 mL × 3), MeOH (3 mL × 3), DMF (3 mL × 3), MeOH (3 mL × 3),

and CH₂Cl₂(3 mL × 3), with E-DOMINO apparatus, and then dried in

reduced pressure to give the desired product.

Figure 7. 36·HCl-induced expression of mRNA related to cartilage

matrix in isolated medial tibial cartilage from MT rats. Aggrecan (a) and

SOX9 (b). The ordinate shows induction ratio (%), mean SE, n = 5

(saline), n = 8 (1 μM), n = 8 (2 μM), n = 7 (5 μM), and n = 8 (10 μM),

(p value *: p < 0.05 **: p < 0.01 ***: p < 0.001, by Dunnett’s test).

Intra-articular injection of 36·HCl-induced chondrogenesis and

promoted repair of cartilage lesions in rats with MT-induced OA.

Rats were subjected to meniscal tear and treatment with either vehicle

alone or vehicle containing 36·HCl, as described under concentration.

Staudinger Reaction Step. A solution of PPh₃(5.0 equiv) in THF

(4.0 mL) was added to the resin in a 10 mL syringe and then agitated at

RT for 16 h. Thereafter, the reaction mixture was washed with each

solvent in the following order: three times of a sequence of THF (3 mL

a

Scheme 1. Polymer-Supported Synthesis of Quinazolin-4(3H)-one Derivatives 4−9, 11−15, 26−29, and 33−36

a

Reagents and conditions: (a) NaNO₂, HCl_(aq); then NaN₃; (b) Wang resin, DIC, HOBt, DIEA, CH₂Cl₂, rt; (c) PPh₃, THF, rt; (d) R₂NCO,

CH₂Cl₂, rt; (e) 43a−d, CH₂Cl₂, rt; (f) MeOH, 60 °C; (g) 1 M HCl in MeOH.

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a

Scheme 2. Synthesis of Quinazolin-4(3H)-one Derivatives 10, 17−19, and 30−32

a

Reagents and conditions: (a) Triphosgene, THF, 0 °C; (b) p-anisidine 46a or 46b, toluene, reﬂux; (c) in the case of 49a−d:

1,1’thiocarbonyldiimidazole, THF, RT or in the case of 49e: CS₂, NaOH, EtOH, reﬂux; (d) SO₂Cl₂, CHCl₃, reﬂux; (e) 43a/e, DMSO, 125 °C.

a

Scheme 3. Synthesis of Quinazolin-4(3H)-one Derivatives 16 and 20−25

a

(a) 43a and 50a−h, DMSO, 125 °C; (b) TFA, CH₂Cl₂, rt; (c) HCHO_(aq), NaBH(OAc)₃, CH₂Cl₂, rt.

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× 3), toluene (3 mL × 3), CH₂Cl₂(3 mL × 3), and hexane (3 mL × 3)

with E-DOMINO apparatus, and then dried in reduced pressure to give

the desired product.

Aza-Wittig Reaction Step. A solution of isocyanate (5.0 equiv) in

CH₂Cl₂(0.3 mL) was added to the resin in a 10 mL syringe and then

agitated at RT for 16 h. Thereafter, the reaction mixture was washed

with each solvent in the following order: two times of a sequence of

THF (3 mL × 3), toluene (3 mL × 3), CH₂Cl₂(3 mL × 3), and hexane

(3 mL × 3) with E-DOMINO apparatus, and then dried in reduced

pressure to give the desired product.

Quinazolin-4(3H)-one-ring Formation Step. A solution of amine

(0.6 equiv) in CH₂Cl₂(0.2 mL) was added to a suspension of the resin

in hexane: CH₂Cl₂= 0.5 mL: 1.5 mL, and the reaction mixture was

agitated at 55 °C for 5 h, then agitated at RT for 16 h. The reaction

mixture was washed with CH₂Cl₂(3 mL × 3) and MeOH (3 mL × 3),

and the organic layer was evaporated with nitrogen blow down and

dried in reduced pressure to give the Quinazolin-3-one (4−9, 11−15,

26−29, 33−36). All compounds’ yields and characteristic data are

shown below.

8-Chloro-3-(4-chlorophenyl)-2-(4-methylpiperazin-1-yl)-

quinazolin-4(3H)-one (4). Yield: 9.6 mg, ¹H NMR (400 MHz,

DMSO-d₆) δ 8.04 (d, 1H, J = 7.5 Hz, Ar), 7.83 (d, 1H, J = 7.5 Hz, Ar),

7.60−7.55 (m, 4H, Ar), 7.50−7.45 (m, 4H, Ar), 7.29 (dd, 1H, J = 7.5,

7.5 Hz, Ar), 3.27 (br t, 4H, J = 5.0 Hz, CH), 2.29 (br t, 4H, J = 5.0 Hz,

CH), 2.24 (s, 3H, NMe); ¹³C NMR (100.6 MHz, CDCl₃) δ 163.05

(CO), 153.85 (ipso-Ar), 144.57 (ipso-Ar), 135.83 (ipso-Ar), 134.67

(Ar), 133.97 (ipso-Ar), 130.26 (Ar), 128.80 (Ar), 125.46 (Ar), 124.34

(Ar), 120.19 (ipso-Ar), 53.49 (CH₂), 48.07 (CH₂), 44.52 (CH₃); MS

(ESI) m/z 390 [(M + H)⁺], RT, 0.96 min; purity, 100% (ELSD), 100%

(PDA).

8-Chloro-2-(4-methylpiperazin-1-yl)-3-(p-tolyl)-quinazolin-

4(3H)-one (5). Yield: 7.2 mg, ¹H NMR (400 MHz, DMSO-d₆) δ 7.94

(dd, 1H, J = 8.0, 1.0 Hz, Ar), 7.87 (dd, 1H, J = 8.0, 1.0 Hz, Ar), 7.36−

7.29 (m, 4H, Ar), 7.26 (dd, 1H, J = 8.0, 8.0 Hz, Ar), 3.12 (dd, 4H, J =

8.0, 8.0 Hz, CH), 2.54 (s, 3H, NMe), 2.38 (s, 3H, Me), 2.11−2.02 (m,

4H, CH); MS (ESI) m/z 370 [(M + H)⁺], RT, 0.92 min; purity, 100%

(ELSD), 100% (PDA).

8-Chloro-2-(4-methylpiperazin-1-yl)-3-(m-tolyl)-quinazolin-

4(3H)-one (6). Yield: 7.8 mg, ¹H NMR (400 MHz, DMSO-d₆) δ 7.94

(dd, 1H, J = 8.0, 1.0 Hz, Ar), 7.87 (dd, 1H, J = 8.0, 1.0 Hz, Ar), 7.40 (dd,

1H, J = 8.0, 8.0 Hz, Ar), 7.30−7.23 (m, 4H, Ar), 3.13 (dd, 4H, J = 8.0,

8.0 Hz, CH), 2.54 (s, 3H, NMe), 2.36 (s, 3H, Me), 2.09−2.00 (m, 4H,

CH); MS (ESI) m/z 370 [(M + H)⁺], RT, 0.93 min; purity, 100%

(ELSD), 100% (PDA).

p-Anisidine 46b (830 mg, 6.74 mmol, 1.1 equiv) was added to a

solution of isatoic anhydride 45d (1.0 g, 6.13 mmol, 1.0 equiv) in

toluene (6.1 mL) and stirred at reﬂux for 4 h. After diluting with hexane

(10 mL), the reaction mixture was ﬁltered out to collect the precipitate.

The precipitate was dried in reduced pressure to give amide 47e (1.43 g,

96%) as a gray solid, with MS (ESI) m/z 243 [(M + H)⁺], RT 1.32 min.

(Step B) 3-(4-Methoxyphenyl)-2-thioxo-2,3-dihydroquinazolin-

4(1H)-one (48e)

A solution of amide 47e (500 mg, 2.06 mmol, 1.0 equiv) in EtOH (4

mL) was added to a solution of NaOH (124 mg, 3.10 mmol, 1.5 equiv)

in CS₂(187 μL, 3.10 mmol, 1.5 equiv), and the reaction mixture was

stirred at reﬂux for 16 h. After cooling to rt, the reaction mixture was

poured to 1 M HCl_(aq)(10 mL) and diluted with hexane (10 mL). The

suspension was ﬁltered to collect the precipitate, and the solid was dried

to give the cyclic compound 48e (679 mg, quant) as a white solid, with

MS (ESI) m/z 285 [(M + H)⁺], RT 1.30 min.

(Step C) 2-Chloro-3-(4-methoxyphenyl)-quinazolin-4(3H)-one

(49e)

Sulfuryl chloride (58 μL, 0.704 mmol, 1.0 equiv) was added to a

solution of thiocarbonyl compound 48e (200 mg, 0.704 mmol, 1.0

equiv) in CHCl₃(1.4 mL) and stirred at reﬂux for 1 h. After cooling to

rt, the reaction mixture was quenched with H₂O (10 mL) and extracted

with CHCl₃(20 mL × 3). The organic layer was combined, dried over

MgSO₄, and evaporated with reduced pressure to give chloride 49e

(139 mg, 69%) as a colorless solid, with MS (ESI) m/z 287 [(M + H)⁺],

RT, 1.56 min.

(Step D) 3-(4-Methoxyphenyl)-2-(4-methylpiperazin-1-yl)-quina-

zolin-4(3H)-one (10)

1-Methylpiperazine 43a was added to a solution of chloride 49e (40

mg, 0.14 mmol, 1.0 equiv) in DMSO (500 μL) and stirred at 125 °C for

1 h. The reaction mixture directly puriﬁed with column chromatog-

raphy on silica with 90:10 CHCl₃/MeOH as eluent gave 10 (16.6 mg,

34%) as a colorless solid, ¹H NMR (400 MHz, DMSO-d₆) δ 8.11 (d,

1H, J = 7.5 Hz, Ar), 7.75 (dd, 1H, J = 7.5, 7.5 Hz, Ar), 7.57 (d, 1H, J =

7.5 Hz, Ar), 7.39−7.33 (m, 3H, Ar), 7.09 (d, 1H, J = 7.5 Hz, Ar), 3.23

(br t, 4H, J = 5.0 Hz, CH), 2.24 (br t, 4H, J = 5.0 Hz, CH), 2.22 (s, 3H,

NMe); ¹³C NMR (100.6 MHz, DMSO-d₆) δ 164.05 (CO), 154.48

(ipso-Ar), 154.48 (ipso-Ar), 147.89 (ipso-Ar), 134.57 (Ar), 129.74 (Ar),

129.62 (ipso-Ar), 126.53 (Ar), 125.66 (Ar), 124.58 (Ar), 118.80 (ipso-

Ar), 113.77 (Ar), 54.63 (OMe), 53.70 (CH₂), 47.84 (CH₂), 44.53

(CH₃); MS (ESI) m/z 352 [(M + 2H)⁺], rt, 0.76 min; purity, 100%

(ELSD), 100% (PDA).

Ethyl 4-(8-Bromo-2-(4-methylpiperazin-1-yl)-4-oxoquinazo-

1

lin-3(4H)-yl)-benzoate (11). Yield: 6.2 mg, H NMR (400 MHz,

8-Chloro-2-(4-methylpiperazin-1-yl)-3-(o-tolyl)-quinazolin-

4(3H)-one (7). Yield: 7.2 mg, ¹H NMR (400 MHz, DMSO-d₆) δ 7.96

(dd, 1H, J = 8.0, 1.0 Hz, Ar), 7.90 (dd, 1H, J = 8.0, 1.0 Hz, Ar), 7.43−

7.33 (m, 4H, Ar), 7.30 (dd, 1H, J = 8.0, 8.0 Hz, Ar), 3.32−3.14 (m, 2H,

CH), 3.08−2.99 (m, 2H, CH), 2.54 (s, 3H, NMe), 2.07−1.96 (m, 4H,

CH). 2.06 (s, 3H, Me); MS (ESI) m/z 370 [(M + H)⁺], RT, 0.94 min;

purity, 100% (ELSD), 99% (PDA).

DMSO-d₆) δ 8.07 (d, 2H, J = 8.5 Hz, Ar), 7.95 (dd, 1H, J = 8.0, 1.5 Hz,

Ar), 7.87 (dd, 1H, J = 8.0, 1.5 Hz, Ar), 7.64 (d, 2H, J = 8.5 Hz, Ar), 7.27

(dd, 1H, J = 8.0, 8.0 Hz, Ar), 4.35 (q, 2H, J = 7.0 Hz, CH₂), 3.14−3.10

(m, 4H, CH), 2.54 (s, 3H, NMe), 2.16−2.13 (m, 4H, CH), 1.35 (t, 3H,

J = 7.0 Hz, CH₃); MS (ESI) m/z 473 [(M + H)⁺], RT, 1.07 min; purity,

100% (ELSD), 97% (PDA).

Ethyl 4-(8-Methoxy-2-(4-methylpiperazin-1-yl)-4-oxoquina-

zolin-3(4H)-yl)-benzoate (12). Yield: 5.4 mg, ¹H NMR (400 MHz,

DMSO-d₆) δ 8.08 (d, 2H, J = 8.5 Hz, Ar), 7.63 (d, 2H, J = 8.5 Hz, Ar),

7.59 (dd, 1H, J = 8.0, 1.5 Hz, Ar), 7.33 (dd, 1H, J = 8.0, 1.5 Hz, Ar), 7.29

(dd, 1H, J = 8.0, 8.0 Hz, Ar), 4.36 (q, 2H, J = 7.0 Hz, CH₂), 3.92 (s, 3H,

OMe), 3.26−3.23 (m, 4H, CH), 3.04−2.99 (m, 4H, CH), 2.54 (s, 3H,

NMe), 1.36 (t, 3H, J = 7.0 Hz, CH₃); MS (ESI) m/z 366 [(M + H)⁺], rt,

0.80 min; purity, 100% (ELSD), 98% (PDA).

Ethyl 4-(8-Chloro-2-(4-methylpiperazin-1-yl)-4-oxoquinazo-

1

lin-3(4H)-yl)-benzoate (8). Yield: 8.5 mg, H NMR (400 MHz,

DMSO-d₆) δ 8.11−8.09 (m, 1H, Ar), 8.09−8.06 (m, 1H, Ar), 7.96 (dd,

1H, J = 8.0, 1.5 Hz, Ar), 7.90 (dd, 1H, J = 8.0, 1.5 Hz, Ar), 7.67−7.65

(m, 1H, Ar), 7.65−7.63 (m, 1H, Ar), 7.29 (dd, 1H, J = 8.0 Hz, Ar), 4.36

(q, 2H, J = 7.0 Hz, CH₂), 3.10 (dd, 4H, J = 4.0, 4.0 Hz, CH), 2.12−2.00

(m, 4H, CH), 2.06 (s, 3H), 1.36 (t, 3H, J = 7.0 Hz, CH₃); MS (ESI) m/

z 428 [(M + H)⁺], RT, 1.01 min; purity, 100% (ELSD), 95% (PDA).

Ethyl 4-(8-Fluoro-2-(4-methylpiperazin-1-yl)-4-oxoquinazo-

Ethyl 4-(8-Fluoro-2-(4-isopropylpiperazin-1-yl)-4-oxoquina-

zolin-3(4H)-yl)-benzoate (13). Yield: 3.0 mg, ¹H NMR (400 MHz,

DMSO-d₆) δ 8.08 (d, 2H, J = 8.5 Hz, Ar), 7.82 (d, 1H, J = 8.0 Hz, Ar),

7.64 (d, 2H, J = 8.5 Hz, Ar), 7.62−7.59 (m, 1H, Ar), 7.34−7.27 (m, 1H,

Ar), 4.35 (q, 2H, J = 7.0 Hz, CH₂), 3.07−3.04 (m, 4H, CH), 2.54 (s,

3H, NMe), 2.16−2.13 (m, 4H, CH), 1.35 (t, 3H, J = 7.0 Hz, CH₃),

1.31−1.23 (m, 1H, CH), 0,85 (d, 6H, J = 7.0 Hz, CH); MS (ESI) m/z

440 [(M + H)⁺], RT, 1.00 min; purity, 98% (ELSD), 95% (PDA).

Ethyl 4-(2-(3-(Dimethylamino)-pyrrolidin-1-yl)-8-ﬂuoro-4-

1

lin-3(4H)-yl)-benzoate (9). Yield: 8.3 mg, H NMR (400 MHz,

DMSO-d₆) δ 8.21 (d, 2H, J = 8.5 Hz, Ar), 7.95 (d, 1H, J = 8.0 Hz, Ar),

7.63 (d, 2H, J = 8.5 Hz, Ar), 7.55 (dd, 1H, J = 8.0, 8.0 Hz, Ar), 7.41−

7.33 (m, 1H, Ar), 4.35 (q, 2H, J = 7.0 Hz, CH₂), 3.33 (br s, 4H, CH),

2.87 (br s, 4H, CH), 2.67 (s, 3H, NMe), 1.35 (t, 3H, J = 7.0 Hz, CH₃;

MS (ESI) m/z 412 [(M + H)⁺], RT, 0.93 min; purity, 100% (ELSD),

95% (PDA).

Synthesis of 3-(4-Methoxyphenyl)-2-(4-methylpiperazin-1-

yl)-quinazolin-4(3H)-one (10). (Step A) 2-Amino-N-(4-methoxy-

phenyl)-benzamide (47e)

1

oxoquinazolin-3(4H)-yl)-benzoate (14). Yield: 4.7 mg, H NMR

(400 MHz, DMSO-d₆) δ 8.14−8.05 (m, 2H, Ar), 7.75 (d, 1H, J = 8.0

Hz, Ar), 7.58−7.50 (m, 3H, Ar), 7.19−7.13 (m, 1H, Ar), 4.36 (q, 2H, J

1477

DOI: 10.1021/acs.jmedchem.8b01615

J. Med. Chem. 2019, 62, 1468−1483

Journal of Medicinal Chemistry

Article

= 7.0 Hz, CH₂), 3.31−3.27 (m, 4H, CH), 3.07−2.98 (m, 1H, CH), 2.00

(s, 6H, NMe), 1,92−1,83 (m, 1H, CH), 1.55−1.42 (m, 1H, CH), 1.35

(t, 3H, J = 7.0 Hz, CH₃); MS (ESI) m/z 426 [(M + H)⁺], RT, 0.97 min;

purity, 98% (ELSD), 95% (PDA).

Ethyl 4-(8-Fluoro-2-(methyl(1-methylpyrrolidin-3-yl)-

amino)-4-oxoquinazolin-3(4H)-yl)-benzoate (15). Yield: 7.8 mg,

¹H NMR (400 MHz, DMSO-d₆) δ 8.14 (d, 2H, J = 8.0 Hz, Ar), 7.79 (d,

2H, J = 8.0, Ar), 7.71 (d, 1H, J = 8.0 Hz, Ar), 7.52−7.48 (m, 1H, Ar),

7.28−7.23 (m, 1H, Ar), 4.36 (q, 2H, J = 7.0 Hz, CH₂), 3.00 (s, 3H,

NMe), 2.82 (d, 1H, J = 1.0 Hz, CH), 2.77 (d, 2H, J = 4.5 Hz, CH),

2.71−2.65 (m, 2H, CH), 2.62 (s, 3H, NMe), 2.17 (m, 2H, CH), 1.35 (t,

3H, J = 7.0 Hz, CH₃); MS (ESI) m/z 426 [(M + H)⁺], RT, 0.91 min;

purity, 100% (ELSD), 96% (PDA).

Synthesis of 7-Fluoro-3-(4-methoxyphenyl)-2-(piperazin-1-

yl)- quinazolin-4(3H)-one (16). (Step A) tert-Butyl 4-(7-ﬂuoro-3-(4-

methoxyphenyl)-4-oxo-3,4-dihydroquinazolin -2-yl)-piperazine-1-car-

boxylate (51a)

tert-Butyl 1-piperazine carboxylate 50b (24.6 mg, 131 μmol, 2.0

equiv) was added to a solution of chloride 49d (20 mg, 66 μmol, 1.0

equiv) in DMSO (500 μL) and stirred at 125 °C for 1 h. The reaction

mixture directly puriﬁed with column chromatography on silica with

90:10 CHCl₃/MeOH as eluent gave 51a (37.7 mg, quant) as a colorless

solid, with MS (ESI) m/z 455 [(M + H)⁺], RT, 1.93 min.

(Step B) 7-Fluoro-3-(4-methoxyphenyl)-2-(piperazin-1-yl)-quina-

zolin-4(3H)-one (16)

The reaction mixture directly puriﬁed with column chromatography on

silica with 90:10 CHCl₃/MeOH as eluent gave 19 (5.5 mg, 16%) as a

yellow oil, ¹H NMR (400 MHz, DMSO-d₆) δ 8.02 (d, 1H, J = 8.0 Hz,

Ar), 7.66 (dd, 1H, J = 8.0, 8.0 Hz, Ar), 7.41 (d, 1H, J = 8.0 Hz, Ar),

7.29−7.25 (m, 2H, Ar), 7.23−7.12 (m, 3H, Ar), 3.57 (br t, 4H, J = 5.0

Hz, CH), 2.68 (br t, 4H, J = 5.0 Hz, CH), 2.39 (s, 6H, NMe); ¹³C NMR

(100.6 MHz, DMSO-d₆) δ 163.53 (CO), 160.71 (ipso-Ar), 149.56

(ipso-Ar), 134.53 (Ar), 129.74 (Ar), 126.81 (ipso-Ar), 126.41 (Ar),

124.28 (Ar), 122.15 (Ar), 116.96 (ipso-Ar), 115.27 (Ar), 57.72 (CH₃),

54.68 (CH₂), 43.76 (CH₃), 38.36 (CH₂); MS (ESI) m/z 423 [(M +

H)⁺], RT, 1.11 min; purity, 100% (ELSD), 100% (PDA).

Synthesis of (S)-7-Fluoro-2-(3-isopropyl-4-methylpiperazin-

1-yl)-3-(4-(triﬂuoromethoxy)-phenyl)-quinazolin-4(3H)-one

(20). (Step A) tert-Butyl (S)-4-(7-ﬂuoro-4-oxo-3-(4-(triﬂuorome-

thoxy)-phenyl)-3,4-dihydro-quinazolin-2-yl)-2-isopropylpiperazine-1-

carboxylate was prepared from 49c with (S)-N-Boc-2-isopropylpiper-

azine 50c analogous to the synthesis of 51a from 49d to aﬀord 51b

(20.8 mg, 46%) as a colorless solid, MS (ESI) m/z 551 [(M + H)⁺], RT,

2.29 min.

(Step B) (S)-7-Fluoro-2-(3-isopropyl-4-methylpiperazin-1-yl)-3-(4-

(triﬂuoromethoxy) -phenyl)-quinazolin-4(3H)-one (20)

HCl_(aq)(35%, 200 μL) was added to a solution of 51b (20.8 mg, 38

μmol, 1.0 equiv) in MeOH (300 μL) and stirred at RT for 1 h. After

evaporating the solvent, the residue was dissolved to 1,2-dichloro-

ethane/AcOH (3:1) (420 μL) and then 35% HCHO_(aq)(160 mL) was

added to the reaction mixture at rt. After stirring for 10 min at rt,

NaBH(OAc)₃(24 mg) was added to the reaction mixture at 0 °C, then

stirred for 16h at rt. The reaction mixture directly puriﬁed with column

chromatography on silica with 100:9:1 CHCl₃/MeOH/NH₄OH as

eluent gave 20 (12.7 mg, 72%) as a colorless solid, ¹H NMR (400 MHz,

DMSO-d₆) δ 8.16 (dd, 1H, J = 8.0, 6.0 Hz, Ar), 7.60 (br s, 2H, Ar), 7.47

(d, 2H, J = 8.0 Hz, Ar), 7.24 (dd, 1H, J = 10.0, 3.0 Hz, Ar), 7.13 (ddd,

1H, J = 10.0, 10.0, 3.0 Hz, Ar), 3.52 (dd, 1H, J = 12.0, 3.0 Hz, CH),

3.36−3.32 (1H, m, CH), 3.04 (ddd, 1H, J = 12.0, 12.0, 3.0 Hz, CH),

2.77 (ddd, 1H, J = 12.0, 3.0, 3.0 Hz, CH), 2.52 (dd, 1H, J = 12.0, 12.0

Hz, CH), 2.18 (s, 3H, NMe), 2.12 (ddd, 1H, J = 12.0, 12.0, 3.0 Hz,

CH), 2.05−1.93 (m, 1H, CH), 1.45 (ddd, 1H, J = 12.0, 3.0, 3.0 Hz,

TFA (95 mg, 0.83 mmol, 10 equiv) was added to a solution of

chloride 51a (37.7 mg, 83 μmol, 1.0 equiv) in CH₂Cl₂(830 μL) and

stirred at RT for 1 h. The reaction mixture was neutralized with

NH₄OH_(aq)and evaporated to give the crude product. Puriﬁcation with

column chromatography on silica with 90:10 CHCl₃/MeOH as eluent

1

gave 16 (37.7 mg, quant) as a colorless solid, H NMR (400 MHz,

DMSO-d₆) δ 8.19 (dd, 1H, J = 8.0, 6.0 Hz, Ar), 7.39 (d, 2H, J = 8.0 Hz,

Ar), 7.25 (dd, 1H, J = 10.0, 3.0 Hz, Ar), 7.19 (ddd, 1H, J = 8.0, 8.0, 2.5

Hz, Ar), 7.12 (d, 2H, J = 8.0, Hz, Ar), 3.43 (br t, 4H, J = 5.0 Hz, CH),

3.04 (br t, 4H, J = 5.0 Hz, CH); MS (ESI) m/z 355 [(M + H)⁺], RT,

0.86 min; purity, 100% (ELSD), 100% (PDA).

Synthesis of 7-Fluoro-3-(4-methoxyphenyl)-2-(4-methylpi-

perazin-1-yl)-quinazolin-4(3H)-one (17). (Step A) 2-Amino-4-

ﬂuoro-N-(4-methoxyphenyl)-benzamide (47d) was prepared from 45c

analogous to the synthesis of 47e from 45d to aﬀord 47d (650 mg,

90%) as a gray solid, with MS (ESI) m/z 261 [(M + H)⁺], RT, 1.41 min.

(Step B) 7-Fluoro-3-(4-methoxyphenyl)-2-thioxo-2,3-dihydroqui-

nazolin-4(1H)-one (48d) was prepared from 47d analogous to the

synthesis of 48e from 47e to aﬀord 48d (251 mg, quant) as a colorless

solid, with MS (ESI) m/z 303 [(M + H)⁺], RT, 1.38 min.

(Step C) 2-Chloro-7-ﬂuoro-3-(4-methoxyphenyl)-quinazolin-

4(3H)-one (49d) was prepared from 48d analogous to the synthesis

of 49e from 48e to aﬀord 49d (66.4 mg, 66%) as a colorless solid, MS

(ESI) m/z 305 [(M + H)⁺], RT, 4.34 min (condition B).

(Step D) 7-Fluoro-3-(4-methoxyphenyl)-2-(4-methylpiperazin-1-

yl)-quinazolin-4(3H)-one (17) was prepared from 49d analogous to

the synthesis of 10 from 49e to aﬀord 17 (23.2 mg, 95%) as a colorless

solid, ¹H NMR (400 MHz, DMSO-d₆) δ 8.14 (dd, 1H, J = 8.0, 6.0 Hz,

Ar), 7.34 (d, 2H, J = 8.0 Hz, Ar), 7.21 (dd, 1H, J = 10.0, 3.0 Hz, Ar), 7.12

(ddd, 1H, J = 8.0, 8.0, 2.5 Hz, Ar), 7.09 (d, 2H, J = 8.0, Hz, Ar), 3.23 (br

t, 4H, J = 5.0 Hz, CH), 2.24 (br t, 4H, J = 5.0 Hz, CH), 2.22 (s, 3H,

NMe); MS (ESI) m/z 423 [(M + H)⁺], RT 1.11 min.

2-(4-Ethylpiperazin-1-yl)-7-ﬂuoro-3-(4-methoxyphenyl)-

quinazolin-4(3H)-one (18). 18 was prepared from 49d with 1-

ethylpiperazine 50a analogous to the synthesis of 10 from 49e to aﬀord

18 (22.1 mg, 88%) as a colorless solid, ¹H NMR (400 MHz, DMSO-d₆)

δ 8.14 (dd, 1H, J = 8.0, 6.0 Hz, Ar), 7.34 (d, 2H, J = 8.0 Hz, Ar), 7.21

(dd, 1H, J = 10.0, 3.0 Hz, Ar), 7.14−7.05 (m, 3H, Ar), 3.24 (br t, 4H, J =

5.0 Hz, CH), 2.36 (q, 2H, J = 7.0 Hz, CH₂), 2.26 (br t, 4H, J = 5.0 Hz,

CH), 1.06 (t, 3H, J = 7.0 Hz, CH); MS (ESI) m/z 423 [(M + H)⁺], RT,

1.11 min; purity, 100% (ELSD), 100% (PDA).

2-((2-(Dimethylamino)-ethyl)amino)-3-(4-methoxyphenyl)-

quinazolin-4(3H)-one (19). N,N′-Dimethylethylenediamine 43e (19

μL, 0.21 mmol, 2.0 equiv) was added to a solution of chloride (40 mg,

0.14 mmol, 1.0 equiv) in DMSO (500 μL) and stirred at 125 °C for 1 h.

CH), 0.80 (d, 1H, J = 7.5 Hz, CH₃), 0.66 (d, 1H, J = 7.5 Hz, CH₃); ¹³

C

NMR (100.6 MHz, DMSO-d₆) δ 162.64 (CO), 155.12 (ipso-Ar),

148.58 (ipso-Ar), 135.87 (Ar), 134.67 (Ar), 130.26 (Ar), 129.67 (Ar),

121.28 (Ar), 113.12 (Ar), 110.62 (ipso-Ar), 66.68 (CH), 55.14 (CH₂),

40.85 (CH₃), 26.17 (CH₂), 18.76 (CH₂), 14.00 (CH₂); MS (ESI) m/z

466 [(M + H)⁺], RT, 1.27 min; purity, 100% (ELSD), 100% (PDA).

Synthesis of (S)-7-Fluoro-2-(3-isobutyl-4-methylpiperazin-

1-yl)-3-(4-(triﬂuoromethoxy)-phenyl)-quinazolin-4(3H)-one

(21). (Step A) tert-butyl (S)-4-(7-ﬂuoro-4-oxo-3-(4-(triﬂuorome-

thoxy)-phenyl)-3,4-dihydro -quinazolin-2-yl)-2-isobutylpiperazine-1-

carboxylate was prepared from 49c with (S)-N-Boc-2-isobutylpiper-

azine 50d analogous to the synthesis of 51a from 49d to aﬀord 51c

(18.4 mg, 39%) as a colorless solid, with MS (ESI) m/z 565 [(M +

H)⁺], RT, 2.38 min.

(Step B) (S)-7-Fluoro-2-(3-isobutyl-4-methylpiperazin-1-yl)-3-(4-

(triﬂuoromethoxy)-phenyl)-quinazolin-4(3H)-one (21) was prepared

from 51c analogous to the synthesis of 51b from 20 to aﬀord 21 (15.9

mg, quant) as a colorless solid, ¹H NMR (400 MHz, DMSO-d₆) δ 8.16

(dd, 1H, J = 8.0, 6.0 Hz, Ar), 7.60 (br s, 2H, Ar), 7.47 (d, 2H, J = 8.0 Hz,

Ar), 7.24 (dd, 1H, J = 10.0, 3.0 Hz, Ar), 7.14 (ddd, 1H, J = 10.0, 10.0, 3.0

Hz, Ar), 3.48 (dd, 1H, J = 12.0, 3.0 Hz, CH), 3.04 (ddd, 1H, J = 12.0,

12.0, 3.0 Hz, CH), 2.73 (ddd, 1H, J = 12.0, 3.0, 3.0 Hz, CH), 2.50 (dd,

1H, J = 12.0, 12.0 Hz, CH), 2.23 (s, 3H, NMe), 2.13 (ddd, 1H, J = 12.0,

12.0, 3.0 Hz, CH), 1.45−1.27 (m, 2H, CH), 1.00 (ddd, 1H, J = 12.0,

10.0, 3.0 Hz, CH), 0.88 (d, 1H, J = 7.5 Hz, CH₃), 0.78 (d, 1H, J = 7.5

Hz, CH₃); ¹³C NMR (100.6 MHz, DMSO-d₆) δ 162.64 (CO),

154.67 (ipso-Ar), 150.11 (ipso-Ar), 148.66 (ipso-Ar), 135.72 (Ar),

129.68 (Ar), 129.57 (Ar), 121.05 (Ar), 115.59 (ipso-Ar), 113.20 (Ar),

112.96 (Ar), 110.88 (Ar), 110.67 (Ar), 60.05 (CH), 54.19 (CH₂),

52.95 (CH₂), 41.02 (CH₃), 38.86 (CH₂), 24.96 (CH₂), 22.85 (CH₃),

20.97 (CH₃); MS (ESI) m/z 480 [(M + H)⁺], RT, 1.38 min; purity,

100% (ELSD), 100% (PDA).

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Synthesis of 7-Fluoro-3-(4-(triﬂuoromethoxy)-phenyl)-2-

(3,3,4-trimethylpiperazin-1-yl)-quinazolin-4(3H)-one (22).

(Step A) tert-Butyl 4-(7-ﬂuoro-4-oxo-3-(4-(triﬂuoromethoxy)-phe-

nyl)-3,4-dihydro-quinazolin-2-yl)-2,2-dimethylpiperazine-1-carboxy-

late was prepared from 49c with 1-Boc-2,2-dimethyl-piperazine 50e

analogous to the synthesis of 51a from 49d to aﬀord 51d (19.2 mg,

43%) as a colorless solid, with MS (ESI) m/z 537 [(M + H)⁺], RT, 2.24

min.

(Step B) 7-Fluoro-3-(4-(triﬂuoromethoxy)-phenyl)-2-(3,3,4-trime-

thylpiperazin-1-yl)-quinazolin-4(3H)-one (22) was prepared from 51d

analogous to the synthesis of 51b from 20 to aﬀord 22 (14.8 mg, 92%)

as a colorless solid, ¹H NMR (400 MHz, DMSO-d₆) δ 8.17 (dd, 1H, J =

8.0, 6.0 Hz, Ar), 7.62−7.56 (m, 2H, Ar), 7.50 (d, 2H, J = 8.0 Hz, Ar),

7.27 (dd, 1H, J = 10.0, 3.0 Hz, Ar), 7.17 (ddd, 1H, J = 10.0, 10.0, 3.0 Hz,

Ar), 3.23 (dd, 2H, J = 12.0, 3.0 Hz, CH), 3.00 (br s, 2H, CH), 2.48 (dd,

1H, J = 6.5, 6.5 Hz, CH), 2.50 (dd, 1H, J = 6.5, 6.5 Hz, CH), 2.28 (s, 3H,

NMe), 1.97 (s, 2H, CH), 0.88 (s, 6H, CH₃); ¹³C NMR (100.6 MHz,

DMSO-d₆) δ 162.75 (CO), 155.25 (ipso-Ar), 150.11 (ipso-Ar),

150.06 (ipso-Ar), 135.85 (Ar), 131.10 (Ar), 129.66 (Ar), 129.55 (Ar),

121.44 (Ar), 115.85 (ipso-Ar), 113.57 (Ar), 113.33 (Ar), 111.13 (Ar),

110.91 (Ar), 59.59 (C), 54.95 (CH₂), 48.72 (CH₂), 48.53 (CH₂),

35.66 (CH₃); MS (ESI) m/z 452 [(M + H)⁺], RT, 1.20 min; purity,

100% (ELSD), 100% (PDA).

Synthesis of 7-Fluoro-2-(4-methyl-4,7-diazaspiro[2.5]octan-

7-yl)-3-(4-(triﬂuoromethoxy)-phenyl)-quinazolin-4(3H)-one

(23). (Step A) tert-Butyl 7-(7-ﬂuoro-4-oxo-3-(4-(triﬂuoromethoxy)-

phenyl)-3,4-dihydro-quinazolin-2-yl)-4,7-diazaspiro[2.5]octane-4-car-

boxylate was prepared from 49c with 4,7-Diaza-spiro[2,5]octane-4-

carboxylic acid tert-butyl ester 50f analogous to the synthesis of 51a

from 49d to aﬀord 51e (18.8 mg, 42%) as a colorless solid, with MS

(ESI) m/z 535 [(M + H)⁺], RT 2.22 min.

(Step B) 7-Fluoro-2-(4-methyl-4,7-diazaspiro[2.5]octan-7-yl)-3-(4-

(triﬂuoromethoxy)-phenyl)-quinazolin-4(3H)-one (23) was prepared

from 51f analogous to the synthesis of 51b from 20 to aﬀord 23 (10.7

mg, 68%) as a colorless solid, ¹H NMR (400 MHz, DMSO-d₆) δ 8.16

(dd, 1H, J = 8.0, 6.0 Hz, Ar), 7.61−7.56 (m, 2H, Ar), 7.48 (d, 2H, J = 8.0

Hz, Ar), 7.24 (dd, 1H, J = 10.0, 3.0 Hz, Ar), 7.14 (ddd, 1H, J = 10.0,

10.0, 3.0 Hz, Ar), 3.26−3.22 (m, 2H, CH), 3.02 (br s, 2H, CH), 2.69−

2.65 (m, 1H, CH), 2.34 (s, 3H, NMe), 0.56 (dd, 2H, J = 5.5, 5.5 Hz,

CH₂), 0.11 (dd, 2H, J = 5.5, 5.5 Hz, CH₂); ¹³C NMR (100.6 MHz,

DMSO-d₆) δ 162.75 (CO), 155.34 (ipso-Ar), 150.33 (ipso-Ar),

148.53 (ipso-Ar), 136.04 (Ar), 130.92 (Ar), 129.66 (Ar), 129.55 (Ar),

121.23 (Ar), 115.72 (ipso-Ar), 113.19 (Ar), 112.95 (Ar), 110.88 (Ar),

110.65 (Ar), 50.45 (CH₂), 50.23 (CH₂), 42.65 (CH₂), 40.60 (CH₃),

35.57 (CH₂), 10.09 (CH₂); MS (ESI) m/z 450 [(M + H)⁺], RT, 1.20

min; purity, 100% (ELSD), 100% (PDA).

Synthesis of (R)-2-(2,4-Dimethylpiperazin-1-yl)-7-ﬂuoro-3-

(4-(triﬂuoromethoxy)-phenyl)-quinazolin-4(3H)-one (24). (Step

A) tert-Butyl (R)-4-(7-ﬂuoro-4-oxo-3-(4-(triﬂuoromethoxy)-phenyl)-

3,4-dihydro quinazolin-2-yl)-3-methylpiperazine-1-carboxylate was

prepared from 49c with (R)-N-Boc-3-methylpiperazine 50g analogous

to the synthesis of 51a from 49d to aﬀord 51f (10.4 mg, 24%) as a

colorless solid, with MS (ESI) m/z 523 [(M + H)⁺], RT, 2.18 min.

(Step B) (R)-2-(2,4-Dimethylpiperazin-1-yl)-7-ﬂuoro-3-(4-(tri-

ﬂuoromethoxy)-phenyl)-quinazolin-4(3H)-one (24) was prepared

from 51f analogous to the synthesis of 51b from 20 to aﬀord 24 (4.9

mg, 57%) as a colorless solid, ¹H NMR (400 MHz, DMSO-d₆) δ 8.18

(dd, 1H, J = 8.0, 6.0 Hz, Ar), 8.13 (dd, 1H, J = 8.0, 6.0 Hz, Ar), 7.56−

7.43 (m, 4H, Ar), 7.26 (dd, 1H, J = 10.0, 2.5 Hz, Ar), 7.17 (ddd, 1H, J =

10.0, 10.0, 2.5 Hz, Ar), 7.05 (ddd, 1H, J = 10.0, 10.0, 2.5 Hz, Ar), 6.97

(dd, 1H, J = 10.0, 2.5 Hz, Ar), 3.68−3.61 (m, 2H, CH), 3.23−3.19 (m,

2H, CH), 2.30−2.22 (m, 2H, CH), 2.18 (s, 3H, NMe), 2.18−2.14 (m,

1H, CH), 1.13 (d, 3H, J = 6.5 Hz, CH₃); ¹³C NMR (100.6 MHz,

DMSO-d₆) δ 162.52 (CO), 154.62 (ipso-Ar), 143.57 (ipso-Ar),

135.82 (ipso-Ar), 134.67 (Ar), 133.87 (ipso-Ar), 130.57 (Ar), 121.36

(Ar) 115.40 (ipso-Ar), 113.53 (Ar), 113.29 (Ar), 111.19 (Ar), 110.73

(Ar), 59.6 (CH), 53.78 (CH₂), 51.53 (CH₂), 44.79 (CH₃), 14.43

(CH₃); MS (ESI) m/z 437 [(M + H)⁺], RT, 1.23 min; purity, 100%

(ELSD), 100% (PDA).

7-Fluoro-2-(4-methyl-3-oxopiperazin-1-yl)-3-(4-(triﬂuoro-

methoxy)-phenyl)-quinazolin-4(3H)-one (25). 25 was prepared

from 49c with 1-methylpiperazin-2-one 50h analogous to the synthesis

of 51a from 49d to aﬀord 25 (23.3 mg, 53%) as a colorless solid, ¹H

NMR (400 MHz, DMSO-d₆) δ 8.16 (dd, 1H, J = 8.0, 6.0 Hz, Ar), 7.61−

7.56 (m, 2H, Ar), 7.49 (d, 2H, J = 8.0 Hz, Ar), 7.25 (dd, 1H, J = 10.0, 3.0

Hz, Ar), 7.15 (ddd, 1H, J = 10.0, 10.0, 3.0 Hz, Ar), 3.84 (br s, 2H, CH),

3.37 (dd, 2H, J = 5.0, 5.0 Hz, CH), 3.06 (dd, 2H, J = 5.0, 5.0 Hz, CH),

2.89 (s, 3H, NMe); ¹³C NMR (100.6 MHz, DMSO-d₆) δ 166.81 (C

O), 162.57 (CO), 153.16 (ipso-Ar), 150,02 (ipso-Ar), 149.89 (ipso-

Ar), 148.78 (Ar), 135.57 (Ar), 130.70 (Ar), 129.67 (Ar), 129.56 (Ar),

121.40 (Ar), 115.70 (ipso-Ar), 113.39 (Ar), 113.16 (Ar), 111.07 (Ar),

110.86 (Ar), 51.15 (CH₂), 45.59 (CH₂), 32.90 (CH₃); MS (ESI) m/z

438 [(M + H)⁺], RT, 1.53 min; purity, 100% (ELSD), 100% (PDA).

8-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-(triﬂuoromethyl)-

1

phenyl)-quinazolin-4(3H)-one (26). Yield: 4.5 mg, H NMR (400

MHz, DMSO-d₆) δ 7.92 (d, 2H, J = 8.5 Hz, Ar), 7.83 (d, 1H, J = 8.0 Hz,

Ar), 7.76 (d, 2H, J = 8.5 Hz, Ar), 7.67−7.61 (m, 1H, Ar), 7.36−7.30 (m,

1H, Ar), 3.05 (dd, 4H, J = 4.5, 4.5 Hz, CH), 2.06−2.00 (m, 4H, CH),

2.05 (s, 3H, NMe); MS (ESI) m/z 408 [(M + H)⁺], RT, 0.97 min;

purity, 96% (ELSD), 96% (PDA).

8-Fluoro-3-(4-isopropylphenyl)-2-(4-methylpiperazin-1-yl)-

1

quinazolin-4(3H)-one (27). Yield: 4.1 mg, H NMR (400 MHz,

DMSO-d₆) δ 7.95 (d, 1H, J = 8.0 Hz, Ar), 7.55 (dd, 1H, J = 9.0 Hz, Ar),

7.48 (d, 2H, J = 8.0 Hz, Ar), 7.40 (d, 2H, J = 8.0 Hz, Ar), 7.40−7.35 (m,

1H, Ar), 3.81 (br s, 4H, CH), 3.08−3.01 (m, 4H, CH), 2.86 (s, 3H,

NMe); MS (ESI) m/z 382 [(M + H)⁺], RT, 1.00 min; purity, 97%

(ELSD), 96% (PDA).

8-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-(triﬂuorome-

thoxy)-phenyl)-quinazolin-4(3H)-one (28). Yield: 4.8 mg, ¹H

NMR (400 MHz, DMSO-d₆) δ 7.92 (d, 1H, J = 8.0 Hz, Ar), 7.63−

7.58 (m, 2H, Ar), 7.55−7.46 (m, 3H, Ar), 7.33 (ddd, 1H, J = 8.0, 8.0, 5.0

Hz, Ar), 3.23 (br t, 4H, J = 5.0 Hz, CH), 2.23 (br t, 4H, J = 5.0 Hz, CH),

2.24 (s, 3H, NMe); ¹³C NMR (100.6 MHz, DMSO-d₆) δ 162.59 (C

O), 157.57 (ipso-Ar), 155.06 (ipso-Ar), 154.12 (ipso-Ar), 148.60 (ipso-

Ar), 137.29 (ipso-Ar), 137.17 (ipso-Ar), 135.90 (Ar), 130.74 (Ar),

124.49 (Ar), 124.42 (Ar), 122.13 (Ar), 122.10 (Ar), 121.19 (Ar),

121.76−129.90−119.88−119.22 (CF₃), 119.69 (ipso-Ar), 53.46

(CH₂), 48.15 (CH₂), 44.48 (CH₃); MS (ESI) m/z 424 [(M + H)⁺],

RT, 1.01 min; purity, 100% (ELSD), 100% (PDA).

8-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-((triﬂuoromethyl)-

thio)-phenyl)-quinazolin-4(3H)-one (29). Yield: 4.0 mg, ¹H NMR

(400 MHz, DMSO-d₆) δ 7.95 (d, 1H, J = 8.0 Hz, Ar), 7.92 (d, 2H, J =

8.0 Hz, Ar), 7.69−7.64 (m, 2H, Ar), 7.59−7.52 (m, 2H, Ar), 7.39 (ddd,

1H, J = 8.0, 8.0, 5.0 Hz, Ar), 3.39 (br s, 4H, CH), 2.92 (br s, 4H, CH),

2.71 (s, 3H, NMe); ¹³C NMR (100.6 MHz, DMSO-d₆) δ 162.12 (C

O), 162.08 (ipso-Ar), 157.62 (ipso-Ar), 155.13 (ipso-Ar), 152.51 (ipso-

Ar), 148.60 (ipso-Ar), 139.31 (ipso-Ar), 136.77 (Ar), 136.66 (Ar),

130.08 (Ar), 125.36 (Ar), 125.29 (Ar), 124.71 (Ar), 122.30 (Ar),

121.10 (Ar), 121.27 (CF₃), 120.16 (ipso-Ar), 119.97 (ipso-Ar), 52.19

(CH₂), 45.83 (CH₂), 42.22 (CH₃); MS (ESI) m/z 440 [(M + H)⁺],

RT, 1.08 min; purity, 100% (ELSD), 95% (PDA).

8-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-((triﬂuoromethyl)-

thio)-phenyl)-quinazolin-4(3H)-one HCl Salt (29·HCl). HCl (1

M) in MeOH (23 μL, 23 μmol, 1.0 equiv) was added to a stirred

solution of 29 (10 mg, 23 μmol, 1.0 equiv) in MeOH (0.5 mL) at RT

and stirred at the same temperature for 10 min. Then, the solvent was

removed in vacuo to give 29·HCl (10.8 mg, 99%) as a colorless solid,

¹H NMR (400 MHz, DMSO-d₆) δ 7.91 (d, 1H, J = 8.0 Hz, Ar), 7.86 (d,

2H, J = 8.0 Hz, Ar), 7.73−7.67 (m, 4H, Ar), 7.41 (ddd, 1H, J = 8.0, 8.0,

5.0 Hz, Ar), 3.60−3.52 (m, 2H, CH), 3.10−3.00 (m, 2H, CH), 2.72−

2.66 (4H, m, CH), 2.51 (s, 3H, NMe).

Synthesis of 5-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-(tri-

ﬂuoromethoxy)-phenyl)-quinazolin-4(3H)-one (30). (Step A)

5-Fluoro-2H-benzo[d][1,3]-oxazine-2,4(1H)-dione (45a)

Triphosgene (1.91 g, 6.45 mmol, 1.0 equiv) was added to a solution

of 6-ﬂuoroanthranilic acid 44a (1.0 g, 6.45 mmol, 1.0 equiv) in THF

(20 mL) and stirred at RT for 4 h. After diluting with hexane (20 mL),

the reaction mixture was ﬁltered out to collect the precipitate. The

precipitate was dried under reduced pressure to give 6-ﬂuoroisatoic

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anhydride 45a (821 mg, 70%) as a colorless solid, with MS (ESI) m/z

180 [(M − H)⁺], RT, 0.82 min.

(Step B) 2-Amino-6-ﬂuoro-N-(4-(triﬂuoromethoxy)-phenyl)-ben-

zamide (47a) was prepared from 45a analogous to the synthesis of 47e

from 45d to aﬀord 47a (175 mg, quant) as a colorless solid, with MS

(ESI) m/z 315 [(M + H)⁺], RT, 1.74 min.

synthesis of 49e from 48e to aﬀord 49c (942 mg, quant) as a colorless

solid, with MS (ESI) m/z 359 [(M + H)⁺], RT, 4.85 min (condition B).

(Step D) 7-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-

(triﬂuoromethoxy)phenyl)quinazolin-4(3H)-one (32) was prepared

from 49c analogous to the synthesis of 10 from 49e to aﬀord 32 (12.9

mg, 50%) as a colorless solid, with ¹H NMR (400 MHz, DMSO-d₆) δ

8.15 (dd, 1H, J = 8.0, 3.0 Hz, Ar), 7.61−7.56 (m, 2H, Ar), 7.49−7.44

(m, 2H, Ar), 7.23 (dd, 1H, J = 10.0, 2.5 Hz, Ar), 7.14 (dd, 1H, J = 8.0,

2.5 Hz, Ar), 3.20 (br t, 4H, J = 5.0 Hz, CH), 2.27−2.15 (m, 4H,

CH&NMe); ¹³C NMR (100.6 MHz, DMSO-d₆) δ 162.68 (CO),

154.85 (ipso-Ar), 150.21 (ipso-Ar), 148.60 (ipso-Ar), 135.93 (Ar),

130.75 (Ar), 129.65 (Ar), 129.54 (Ar), 121.19 (Ar), 115.60 (Ar),

113.17 (Ar), 112.93 (Ar), 110.87 (Ar), 110.65 (Ar), 53.48 (CH₂),

48.15 (CH₂), 44.48 (CH₃); MS (ESI) m/z 423 [(M + H)⁺], RT, 1.11

min; purity, 100% (ELSD), 100% (PDA).

8-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-((tetrahydro-2H-

pyran-4-yl)oxy)-phenyl)-quinazolin-4(3H)-one (33). Yield: 4.2

mg, ¹H NMR (400 MHz, DMSO-d₆) δ 7.94−7.15 (m, 1H, Ar), 7.55−

7.49 (m, 1H, Ar), 7.39−7.35 (m, 2H, Ar), 7.23−7.18 (m, 1H, Ar),

7.16−7.12 (m, 2H, Ar), 4.01−3.93 (m, 4H, CH), 3.66−3.57 (m, 4H,

CH), 3.25−3.17 (m, 1H, CH), 2.84 (s, 3H, NMe), 2.11−2.02 (m, 4H,

CH), 1.81−1.68 (m, 4H, CH); MS (ESI) m/z 440 [(M + H)⁺], RT,

0.88 min; purity, 100% (ELSD), 95% (PDA).

(Step C) 5-Fluoro-2-thioxo-3-(4-(triﬂuoromethoxy)-phenyl)-2,3-

dihydroquinazolin -4(1H)-one (48a) was prepared from 47a analogous

to the synthesis of 48e from 47e to aﬀord 48a (85.3 mg, 94%) as a

colorless solid, with MS (ESI) m/z 357 [(M + H)⁺], RT, 1.59 min.

(Step D) 2-Chloro-5-ﬂuoro-3-(4-(triﬂuoromethoxy)-phenyl)-qui-

nazolin-4(3H)-one (49a) was prepared from 48a analogous to the

synthesis of 49e from 48e to aﬀord 49a (61.3 mg, 71%) as a colorless

solid, with MS (ESI) m/z 359 [(M + H)⁺], RT, 1.77 min.

(Step E) 5-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-(triﬂuorome-

thoxy)-phenyl)-quinazolin-4(3H)-one (30) was prepared from 49a

analogous to the synthesis of 10 from 49e to aﬀord 30 (29.3 mg, quant)

as a colorless solid, ¹H NMR (400 MHz, DMSO-d₆) δ 7.70 (ddd, 1H, J

= 8.0, 8.0, 5.5 Hz, Ar), 7.60−7.55 (m, 2H, Ar), 7.47 (d, 1H, J = 8.0 Hz,

Ar), 7.37 (d, 1H, J = 8.0 Hz, Ar), 7.03 (dd, 1H, J = 10.0, 8.0 Hz, Ar), 3.19

(br t, 4H, J = 5.0 Hz, CH), 2.28−2.17 (m, 7H, CH&NMe); ¹³C NMR

(100.6 MHz, DMSO-d₆) δ 162.95 (CO), 160.33 (ipso-Ar), 154.47

(Ar), 149.95 (Ar), 148.58 (Ar), 135.75 (Ar), 135.22 (ipso-Ar), 135.11

(ipso-Ar), 130.88 (Ar), 121.78 (Ar), 121.75 (Ar), 121.16 (Ar), 110.03

(ipso-Ar), 110.83 (ipso-Ar), 53.49 (CH₂), 48.07 (CH₂), 44.49 (CH₃);

MS (ESI) m/z 423 [(M + H)⁺], RT, 1.04 min; purity, 100% (ELSD),

100% (PDA).

Synthesis of 6-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-(tri-

ﬂuoromethoxy)-phenyl)-quinazolin-4(3H)-one (31). (Step A)

6-Fluoro-2H-benzo[d][1,3]-oxazine-2,4(1H)-dione (45b) was pre-

pared from 45b analogous to the synthesis of 45a from 44a to aﬀord

45b (835 mg, 71%) as a colorless solid, with MS (ESI) m/z 180 [(M −

H)⁺], RT, 2.58 min (condition B).

(Step B) 2-Amino-5-ﬂuoro-N-(4-(triﬂuoromethoxy)-phenyl)-ben-

zamide (47b) was prepared from 45b analogous to the synthesis of 47e

from 45d to aﬀord 47b (193 mg, quant) as a colorless solid, with MS

(ESI) m/z 315 [(M + H)⁺], RT, 1.72 min.

3-(4-Benzylphenyl)-8-ﬂuoro-2-(4-methylpiperazin-1-yl)-qui-

nazolin-4(3H)-one (34). Yield: 4.1 mg, ¹H NMR (400 MHz, DMSO-

d₆) δ 7.94 (d, 1H, J = 8.0 Hz, Ar), 7.54 (dd, 2H, J = 8.0, 8.0 Hz, Ar), 7.45

(d, 2H, J = 8.0 Hz, Ar), 7.42−7.20 (m, 9H, Ar), 7.59−7.52 (m, 2H, Ar),

7.39 (ddd, 1H, J = 8.0, 8.0, 5.0 Hz, Ar), 4.09 (br s, 2H, CH₂), 3.79 (br s,

4H, CH), 3.10 (br s, 4H, CH), 2.82 (s, 3H, NMe); MS (ESI) m/z 430

[(M + H)⁺], RT 1.10 min; purity, 100% (ELSD), 100% (PDA).

8-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-phenoxyphenyl)-

1

quinazolin-4(3H)-one (35). Yield: 3.3 mg, H NMR (400 MHz,

DMSO-d₆) δ 7.86 (d, 1H, J = 1.5 Hz, Ar), 7.64 (dd, 1H, J = 1.5, 1.5 Hz,

Ar), 7.52 (d, 2H, J = 8.0 Hz, Ar), 7.45 (dd, 2H, J = 8.0, 8.0 Hz, Ar),

7.39−7.31 (m, 1H, Ar), 7.23−7.06 (m, 6H, Ar), 6.52 (s, 1H, Ar), 3.31−

3.27 (m, 4H, CH), 2.54 (s, 3H, NMe), 2.51−2.47 (m, 4H, CH); MS

(ESI) m/z 431 [(M + H)⁺], RT, 1.14 min; purity, 97% (ELSD), 96%

(PDA).

8-Fluoro-3-(4-(4-ﬂuorophenoxy)-phenyl)-2-(4-methylpiper-

azin-1-yl)-quinazolin-4(3H)-one (36). Yield: 9.6 mg, ¹H NMR (400

MHz, DMSO-d₆) δ 7.91 (d, 1H, J = 8.0 Hz, Ar), 7.51 (dd, 1H, J = 8.0,

8.0 Hz, Ar), 7.46−7.40 (m, 2H, Ar), 7.35−7.29 (m, 1H, Ar), 7.20−7.09

(m, 6H, Ar), 3.26 (dd, 4H, J = 5.0, 5.0 Hz, CH), 2.28 (dd, 4H, J = 5.0,

5.0 Hz, CH), 2.25 (s, 3H, NMe); ¹³C NMR (100.6 MHz, DMSO-d₆) δ

164.19 (CO), 162.19 (CO), 158.46 (ipso-Ar), 158.25 (ipso-Ar),

133.37 (ipso-Ar), 131.75 (Ar), 124.74 (ipso-Ar), 123.61 (Ar), 123.52

(Ar), 122.36 (Ar), 122.24 (Ar), 121.25 (Ar), 121.13 (Ar), 121.06 (Ar),

119.48 (Ar), 117.74 (Ar), 117.51 (Ar), 55.14 (CH₂), 49.66 (CH₂),

46.07 (NMe); MS (ESI) m/z 449 [(M + H)⁺], RT, 1.20 min; purity,

100% (ELSD), 100% (PDA).

(Step C) 6-Fluoro-2-thioxo-3-(4-(triﬂuoromethoxy)-phenyl)-2,3-

dihydroquinazolin -4(1H)-one (48b) was prepared from 47b

analogous to the synthesis of 48e from 47e to aﬀord 48b (36.2 mg,

40%) as a colorless solid, with MS (ESI) m/z 357 [(M + H)⁺], RT, 1.65

min.

(Step D) 2-Chloro-6-ﬂuoro-3-(4-(triﬂuoromethoxy)-phenyl)-qui-

nazolin-4(3H)-one (49b) was prepared from 48b analogous to the

synthesis of 49e from 48e to aﬀord 49b (28.6 mg, 79%) as a colorless

solid, with MS (ESI) m/z 359 [(M + H)⁺], RT, 4.91 min (condition B).

(Step E) 6-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-(triﬂuorome-

thoxy)-phenyl)-quinazolin-4(3H)-one (31) was prepared from 49b

analogous to the synthesis of 10 from 49e to aﬀord 31 (18.6 mg, quant)

as a colorless solid, ¹H NMR (400 MHz, DMSO-d₆) δ 7.75 (dd, 1H, J =

8.0, 3.0 Hz, Ar), 7.65−7.52 (m, 4H, Ar), 7.49−7.44 (m, 2H, Ar), 3.17

(br t, 4H, J = 5.0 Hz, CH), 2.27−2.15 (m, 4H, CH&NMe); ¹³C NMR

(100.6 MHz, DMSO-d₆) δ 162.71 (CO), 158.69 (ipso-Ar), 153.43

(ipso-Ar), 148.82 (ipso-Ar), 144.54 (Ar), 135.82 (Ar), 130.78 (Ar),

128.39 (Ar), 128.31 (Ar), 123.07 (Ar), 122.83 (Ar), 121.15 (Ar),

111.16 (Ar), 110.93 (Ar), 53.52 (CH₂), 48.29 (CH₂), 44.49 (CH₃);

MS (ESI) m/z 423 [(M + H)⁺], RT, 1.11 min; purity, 100% (ELSD),

100% (PDA).

8-Fluoro-3-(4-(4-ﬂuorophenoxy)-phenyl)-2-(4-methylpiper-

azin-1-yl)-quinazolin-4(3H)-one HCl Salt (36·HCl). 36·HCl was

prepared from 36 analogous to the synthesis of 29·HCl from 29,

1

aﬀording 36·HCl (10.8 mg, 99%) as a colorless solid, H NMR (400

MHz, DMSO-d₆) δ 7.84 (d, 1H, J = 8.0 Hz, Ar), 7.67−7.62 (m, 1H, Ar),

7.54−7.47 (m, 2H, Ar), 7.38−7.25 (m, 3H, Ar), 7.18−7.10 (m, 4H,

Ar), 2.68−2.65 (m, 4H, CH), 2.33−2.31 (m, 4H, CH), 2.30 (s, 3H,

NMe).

Preparation of Azides (38a, 38b, 38c, and 38d) for Solid-

Phase Synthesis. 2-Azido-3-chlorobenzoic Acid (38a). NaNO₂

(1.71 g, 26.4 mmol, 2.2 equiv) in water (10 mL) was added dropwise

to a stirred solution of 2-amino-3-chlorobenzoic acid 37a (2.06 g, 12

mmol, 1.0 equiv) in 6 M HCl_(aq)at 5 °C. After stirring at 0 °C for 4 h,

the solvent was ﬁltered out with a glass funnel to give the yellow solid

and then the yellow solid was dissolved to NaOAc (23.6 g, 288 mmol,

24 equiv) in water (26 mL). NaN₃(1.72 g, 26.4 mmol, 2.2 equiv) in

water (4 mL) was added to a stirred solution. After stirring at 0 °C for 4

h, the resulting mixture was quenched with concentrated HCl_(aq)(5

mL) and the solvent was ﬁltered out. The yellow solid was washed with

6 M HCl_(aq)and dried in reduced pressure, to give the azide product

Synthesis of 7-Fluoro-2-(4-methylpiperazin-1-yl)-3-(4-(tri-

ﬂuoromethoxy)-phenyl)-quinazolin-4(3H)-one (32). (Step A)

2-Amino-4-ﬂuoro-N-(4-(triﬂuoromethoxy)phenyl)-benzamide (47c)

was prepared from 45c analogous to the synthesis of 47e from 45d

to aﬀord 47c (3.0 g, 89%) as a gray solid, with MS (ESI) m/z 315 [(M +

H)⁺], RT, 4.69 min (condition B).

(Step B) 7-Fluoro-2-thioxo-3-(4-(triﬂuoromethoxy)-phenyl)-2,3-

dihydroquinazolin-4(1H)-one (48c) was prepared from 47c analogous

to the synthesis of 48e from 47e to aﬀord 48c (1.69 g, quant) as a

colorless solid, with MS (ESI) m/z 355 [(M + H)⁺], RT, 1.65 min.

(Step C) 2-Chloro-7-ﬂuoro-3-(4-(triﬂuoromethoxy)-phenyl)-qui-

nazolin-4(3H)-one (49c) was prepared from 48c analogous to the

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38a (2.39 g, 28 %) as a yellow solid, ¹H NMR (400 MHz, DMSO-d₆) δ

8.00 (dd, 1H, J = 9.0, 3.0 Hz, Ar), 7.63 (dd, 1H, J = 6.0, 3.0 Hz, Ar), 7.26

(dd, 1H, J = 9.0, 3.0 Hz, Ar); MS (ESI) m/z 196 [(M − H)⁺], RT, 1.26

min.

2-Azido-3-ﬂuorobenzoic Acid (38b). 38b was prepared from 37b

analogous to the synthesis of 38a from 37a to aﬀord 38b (1.17 g, 98 %)

as a brown solid, ¹H NMR (400 MHz, DMSO-d₆) δ 7.89 (ddd, 1H, J =

9.0, 3.0, 3.0 Hz, Ar), 7.39−7.32 (m, 1H, Ar), 7.26−7.18 (m, 1H, Ar);

MS (ESI) m/z 180 [(M − H)⁺], RT, 1.09 min.

2-Azido-3-bromobenzoic Acid (38c). 38c was prepared from 37c

analogous to the synthesis of 38a from 37a to aﬀord 38c (2.39 g, 28 %)

as a yellow solid, ¹H NMR (400 MHz, DMSO-d₆) δ 8.04 (dd, 1H, J =

9.0, 3.0 Hz, Ar), 7.82 (dd, 1H, J = 6.0, 3.0 Hz, Ar), 7.18 (dd, 1H, J = 9.0,

9.0 Hz, Ar); MS (ESI) m/z 227 [(M − H)⁺], RT, 0.27 min.

2-Azido-3-methoxybenzoic Acid (38d). 38d was prepared from 37d

analogous to the synthesis of 38a from 37a to aﬀord 38d (2.39 g, 28 %)

as a yellow solid, ¹H NMR (400 MHz, DMSO-d₆) δ 7.78 (dd, 1H, J =

9.0, 3.0 Hz, Ar), 7.21 (dd, 1H, J = 9.0, 9.0 Hz, Ar), 7.10 (dd, 1H, J = 9.0,

3.0 Hz, Ar); MS (ESI) m/z 192 [(M − H)⁺], RT, 1.08 min.

SOX9 Reporter Gene Assay. The SOX9 reporter gene was

previously reported.¹⁷Human TRPV4 cDNA (NM_021625) was

subcloned into the pcDNA3.1 vector. Reporter assays were performed

using the stable ATDC5 cell line carrying reporter gene constructs and

human TRPV4 expression constructs. Stable cells were inoculated at a

density of 2.0 × 10⁴cells/well in 96-well white plates and treated with

an appropriate concentration of compounds. Luciferase activity was

measured the next day using a Picagene LT 2.0 luminescence kit (Toyo

Ink).

solutions, each joint was embedded in paraﬃn, sliced thinly (5 μm),

double stained with safranine-O and Fast Green, and then observed

under a microscope. Tibial cartilage histopathological evaluation was

used to measure cartilage degeneration in three equally spaced regions

across the surface of the medial tibial plateau. The score is reported as

follows. The term “minimal change” indicates the loss of chondrocytes

and proteoglycans with or without ﬁbrillation involving the superﬁcial

zone. The term “mild change” means the loss of chondrocytes and

proteoglycans with or without ﬁbrillation involving the upper 1/3 of the

cartilage thickness. The term “moderate change” means the loss of

chondrocytes and proteoglycans, with ﬁbrillation extending well into

the mid zone and generally aﬀecting 1/2 of the total cartilage thickness.

The term “marked degeneration” means the loss of chondrocytes and

proteoglycans, with ﬁbrillation extending into the deep zone but with

residual matrix above the tidemark. Finally, the term “severe

degeneration” means matrix loss to the tidemark. The scoring method

allowed strict attention to the zones (outside, middle, and inside 1/3),

and the summed scores reﬂected the global severity of tibial cartilage

degeneration. Signiﬁcant changes in the width of the cartilage were

determined by micrometer measurements of the width of the surface of

the tibial plateau. Chondrocyte and matrix losses were evident in more

than 50% of the cartilage thickness. The depth ratio, which is the

percent of cartilage thickness eroded by a lesion, was determined by

micrometer measurements of the depth of all lesions at four equally

spaced points on the tibial surface. These measurements were taken at

the matrix adjacent to the chondrophyte and at 1/4, 1/2, and 3/4 of the

distance across the tibial plateau. The depth to the tidemark was used to

express cartilage thickness across the tibial plateau. Chondrophyte size

was measured using an ocular micrometer, and the values were averaged

for the three sections taken per animal. Bone and calciﬁed cartilage

damage were used to evaluate damage to the calciﬁed cartilage and

subchondral bone, categorized into six stages:

Measurement of Intracellular Ca²⁺. Cellular Ca²⁺was estimated

using the ratiometric ﬂuorescence Ca²⁺indicator Fura-2. HEK293 cells

transiently expressing human TRPV4 were incubated at 37 °C for 30

min in assay buﬀer (20 mM HEPES (pH 7.4), 115 mM NaCl, 5.4 mM

KCl, 0.8 mM MgSO₄, 1.8 mM CaCl₂, 13.8 mM glucose, and 0.1%

bovine serum albumin), containing 5 μM Fura-2 AM (Dojindo) and

0.2% Pluronic F-127 (molecular probes). The cells were then washed

and resuspended in assay buﬀer. Cellular Ca²⁺was measured by ratio

imaging of Fura-2 ﬂuorescence (emission at 510 nm with excitation at

340 and 380 nm) using a Functional Drug Screening System 3000

(Hamamatsu Pho-tonics).

Normal Human Articular Chondrocyte Cell System Assay.

NHAC cells were obtained from Lonza. We evaluated chondrogenesis

activity by stimulating the cells for 30 min with TRPV4 agonists, then

replacing the medium. After 3 days’ culture, the cells were ﬁxed with

95% methanol and stained with 0.1% Alcian blue 8GS (Sigma) in 0.1 N

HCl overnight. Alcian blue-stained cultures were extracted with 6 M

guanidine−HCl for 2 h at room temperature. The optical density of the

extracted dye was measured at 600 nm.

MT Model and Drug Treatment. Eight-week-old Sprague Dawley

male rats obtained from Japan SLC were housed one per cage and

acclaimed prior to use. All animals were allowed access to food and

water ad libitum before and after surgery. The experimental protocols

were performed according to relevant national and international

guidelines and were approved by the Animal Experimental Ethical

Committee. When the rats were 100 weeks old, they were anesthetized

with pentobarbital and the right knee was prepared for surgery. A skin

incision was made over the medial aspect of the right knee; then, the

medial collateral ligament was exposed by blunt dissection and

transected. A full-thickness cut was made through the medial meniscus

to simulate a complete tear. The muscle and skin were closed with 6-O

Vicryl and 4-O Silk suture, respectively. One week after the surgery, the

rats were divided into four equal-sized groups by body weight: 1, sham

(vehicle); 2, MT (vehicle); 3, MT (5 μM); and 4, MT (10 μM). Next,

50 μL of saline as the vehicle or 36·HCl solution was injected intra-

articularly into the operated knee twice a week for three consecutive

weeks. The rats were euthanized at week 4, and the right knee joints

were harvested for histological evaluation.

1 No change.

2 Increased basophilia at the tidemark but no fragmentation of the

tidemark and no changes to the marrow.

3 Increased basophilia at the tidemark, with minimal to mild

fragmentation of the calciﬁed cartilage.

4 Increased basophilia at the tidemark, with mesenchymal changes

that involve up to 3/4 of the total area. In addition, areas of

chondrogenesis may be evident but there is no collapse of

articular cartilage into the epiphyseal bone.

5 Increased basophilia at the tidemark, with mark to severe

fragmentation of the calciﬁed cartilage and mesenchymal

changes in the marrow that involve up to 3/4 of the total area.

The articular cartilage collapses into the epiphysis to a depth of

250 mm from the tidemark.

6 Increased basophilia at the tidemark with mark to severe

fragmentation of the calciﬁed cartilage and mesenchymal

changes in the marrow that involve up to 3/4 of the total area.

The articular cartilage collapses into the epiphysis to a depth 250

mm from the tidemark.

RNA Isolation and Quantitative Real-Time PCR Analysis.

Cartilage tissue was freeze-pulverized in liquid nitrogen followed by

RNA extraction using an RNAeasy Lipid Tissue Mini kit (Qiagen) with

DNase I (Qiagen) treatment. Total RNA was used to synthesize cDNA

using SuperScript III reverse transcriptase (Invitrogen). Quantitative

real-time PCR was performed on an ABI Prism 7000 Sequence

Detection system using TaqMan Gene Expression Master Mix and

TaqMan Gene Expression Assays (Applied Biosystems). Expression

values were normalized to ribosomal protein RPL19.

Statistical Analysis. All quantitative data are presented as mean

standard deviation (SD). Statistical analyses were performed using

EXSUS ver. 8.1 (CAC Croit) based on SAS ver. 9.2 (SAS Institute

Japan). Comparisons between group-administered vehicle or 5 μM or

10 μM 36·HCl were performed using Dunnett’s test, with the control

group as a reference. Diﬀerences were considered statistically signiﬁcant

at p < 0.05.

Histology and Evaluation. The knee joints were ﬁxed with 10%

neutral buﬀered formalin and decalciﬁed with 5% formic acid formalin.

The decalciﬁed joint was cut to allow observation of the inside and

outside articular surface. After dehydration using graded alcohol

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

■

Corresponding Author

*E-mail: atobe.mb@om.asahi-kasei.co.jp. Phone: +81-558-76-

8493. Fax: +81-558-76-5755.

ORCID

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Masakazu Atobe: 0000-0002-7314-0237

Present Address

^⊥Asahi Kasei Pharma, Diagnostic Department R&D Group,

632-1, Mifuku, Izunokuni, Shizuka, Tokyo 410-2321, Japan

(S.M., M.K.). Asahi Kasei Pharma, Open Innovation Depart-

ment, Hibiya Mitsui Tower, 1-1-2 Yurakucho, Chiyoda-ku,

Tokyo 101-0006, Japan (T.O.).

Author Contributions

^∇M.A. and T.N. contributed equally to this work. The

manuscript was written through contributions of all authors.

All authors have given approval to the ﬁnal version of the

manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors thank Dr. Kenji Takenuki, Yukio Tsuchiya, and

Noritsugu Kitahara from the Laboratory for Medicinal

Chemistry at Asahi Kasei Pharma Corporation, for conducting

the initial chemistry experiments, Dr. Daisuke Shimosato in the

Laboratory for Pharmacology at Asahi Kasei Pharma Corpo-

ration, for conducting the molecular biology experiments, Toru

Kobayashi in the Laboratory for Pharmacology at Asahi Kasei

Pharma Corporation, for supporting in vivo experiments, Eiichi

Tanaka, Satoshi Shiojiri, and Dr. Akio Matsuda in the

Laboratory for Pharmacology at Asahi Kasei Pharma Corpo-

ration, for project management support, and Dr. Shunsuke

Tahara in the Laboratory for Pharmacology at Asahi Kasei

Pharma Corporation, for statistical analysis support.

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ABBREVIATIONS

■

ATDC, a murine chondrogenic cell line; DIC, N,N′-diisopropyl

carbodiimide; DIEA, diisopropyl ethylamine; DMEM, Dulbec-

co’s modiﬁed Eagle medium; DMSO, dimethyl sulfoxide;

EtOH, ethanol; FGF18, ﬁbroblast growth factor 18; GSK,

Glaxo Smith Klein; HEPES, 4-(2-hydroxyethyl)-1-piperazinee-

thanesulfonic acid; HTS, high-throughput screening; MT,

medial meniscal tear; NHAC, normal human articular

chondrocyte; OA, osteoarthritis; 4α-PDD, 4α-phorbol-12,13-

didecanoate; PK, pharmacokinetic; POA, primary osteoarthritis;

PTOA, post-traumatic osteoarthritis; SAR, structure−activity

relationship; SOX, SRY (sex-related Y)-type high-mobility

group box; TFA, triﬂuoroacetic acid; THF, tetrahydrofuran;

TRPV4, transient receptor potential vanilloid 4

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DOI: 10.1021/acs.jmedchem.8b01615

J. Med. Chem. 2019, 62, 1468−1483

Article Doi

Discovery of Novel Transient Receptor Potential Vanilloid 4 (TRPV4) Agonists as Regulators of Chondrogenic Differentiation: Identification of Quinazolin-4(3 H)-ones and in Vivo Studies on a Surgically Induced Rat Model of Osteoarthritis

DOI: 10.1021/acs.jmedchem.8b01615

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Article abstract of DOI:10.1021/acs.jmedchem.8b01615

Full text of DOI:10.1021/acs.jmedchem.8b01615

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