Discovery and Development of S6821 and S7958 as Potent TAS2R8 Antagonists

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Discovery and Development of S6821 and S7958 as Potent TAS2R8

Antagonists

Joseph R. Fotsing,* Vincent Darmohusodo, Andrew P. Patron, Brett W. Ching, Thomas Brady,

Melissa Arellano, Qing Chen, Timothy J. Davis, Hanghui Liu, Guy Servant, Lan Zhang, Mark Williams,

Michael Saganich, Tanya Ditschun, Catherine Tachdjian, and Donald S. Karanewsky

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ABSTRACT: In humans, bitter taste is mediated by 25 TAS2Rs.

Many compounds, including certain active pharmaceutical

ingredients, excipients, and nutraceuticals, impart their bitter

taste (or in part) through TAS2R8 activation. However, eﬀective

TAS2R8 blockers that can either suppress or reduce the bitterness

of these compounds have not been described. We are hereby

reporting a series of novel 3-(pyrazol-4-yl) imidazolidine-2,4-

diones as potent and selective TAS2R8 antagonists. In human

sensory tests, S6821 and S7958, two of the most potent analogues

from the series, demonstrated eﬃcacy in blocking TAS2R8-

mediated bitterness and were selected for development. Following

data evaluation by expert panels of a number of national and

multinational regulatory bodies, including the US, the EU, and Japan, S6821 and S7958 were approved as safe under conditions of

intended use as bitter taste blockers.

salt taste perception in rodents, ENaC has been shown to

participate, at least partially, but it does not seem to play a

signiﬁcant role in human salt taste perception.¹⁴Other

candidate salt receptors are still under evaluation.

INTRODUCTION

■

Taste and taste modulation research has long been driven by

sensory evaluation with the tongue as the sole testing tool. It

was not until recently, with the discovery and characterization

of many vertebrate taste receptors,¹⁻³that taste research has

evolved to using high-throughput screening assays and

medicinal chemistry tools to identify and optimize ligands

(agonists, antagonists, and positive allosteric modulators) that

interact with taste receptors to impart speciﬁc taste modalities

(Figure 1).^1,2,4−9Tremendous progress has been made toward

the understanding of the taste signaling mechanism^1,2,4,8−12

and the development of assays to evaluate the response of taste

receptors to various ligands. Umami and sweet tastes are

mediated by TAS1R1/TAS1R3 and TAS1R2/TAS1R3,

respectively, which are heterodimeric G-protein coupled

receptors (GPCRs).¹³Sour and salty tastes, for their part,

are believed to be mediated by ion channels.^8,9,12,14Early

research suggested that the dimeric complex PKD1L3/

PKD2L1, a member of the TRP family of ion channels, is a

potential candidate receptor for sour taste.¹⁵However, Horio

et al. later demonstrated that PKDs only contribute partly to

the detection of sour taste in mice, thus pointing at the

involvement of other receptors in sour taste mediation.¹⁶More

recently, the work by Tu et al. identiﬁed otopetrin1 (OTOP1),

a proton permeable protein that also plays a key role in the

formation of gravity-sensing calcium carbonate crystals in the

utricle of the ear, as being the sour taste receptor.^8,9,17−19For

Strikingly, bitter taste is mediated by a large family of

GPCRs called TAS2Rs (or T2Rs).²⁰⁻²⁴A single TAS2R can

recognize a diverse variety of bitter ligands, and a bitter

compound can activate multiple TAS2Rs.^5,7,25,26Bitter taste

receptors are present throughout the body and believed to be

also implicated in nontaste related roles, including the

mediation of many human health conditions and diseases.²⁷⁻³⁸

Humans have 25 full-length TAS2Rs, which are highly

divergent in sequence, sharing only 30−70% amino acid

homology.³⁹Additionally, there are more than 150 single

nucleotide polymorphisms among individual TAS2R

genes,^3,40−43several of which are responsible for variation in

the liking and in the intensity of human bitter taste perception

of various bitter tastants.⁴³⁻⁵⁵For TAS2R8, there are 5 known

variants causing amino acid changes.^40,56Those variants add

more diversity to the already complex machinery of bitter taste

Received: March 5, 2020

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Figure 1. Taste receptors and examples of ligands or tastants.^1,2,4,5,8,9

perception that has been built over millions of years of

evolution and is believed to be a means of selecting safe-to-

ingest nutrients; however, a clear correlation between bitter-

ness and toxicity has not yet been established.⁵⁷In fact, while

many toxic compounds are known to impart a bitter taste

response,⁵⁸⁻⁶⁰many medicines and compounds with poten-

tially beneﬁcial properties have also been described as

exhibiting a bitter taste.^6,7,61−63Thus, the development of

bitter taste modulators is essential not only as a means to

generate tools for studying the physiological function of bitter

taste receptors but also as an approach to improve the

palatability of foods, beverages, and ingestible pharmaceuticals

associated with bitter taste. Despite nutrition and health

beneﬁts, the bitterness of certain foods, beverages, and drug

formulations is the cause of a high rate of noncompliance by

consumers and patients. In the case of drugs such as

antibiotics, noncompliance may lead to the development of

resistance, the worsening of symptoms, and an increase in

treatment cost. Although the use of sweeteners and taste-

masking techniques have been helpful in alleviating the

bitterness associated with certain active pharmaceutical

ingredients (APIs) and consumable goods, these techniques

are often limited to certain applications or come with their own

palatability issues. For example, coating techniques cannot be

used for liquid drugs, some popular sweeteners (e.g., RebA)

are known to have a bitter oﬀ-taste themselves, and some are

simply incompatible with certain formulations. Thus, there is

still a pressing need for new APIs for bitter taste blockers.^6,64,65

Moreover, many consumers ﬁnd coﬀee too bitter, adding many

grams of sugar to enhance palatability. With that in mind, we

sought to leverage our bitter taste receptor technology

platform to develop bitter taste receptor antagonists as

bitterness modulation alternatives that could eﬀectively

alleviate the bitterness imparted by coﬀee, beverages, foods,

and APIs.

25 TAS2Rs have been reported to date.^5,25,66−74Herein, we

describe the discovery and optimization of a new class of bitter

antagonists typiﬁed by S6821 and S7958. These two

compounds block the activation of the human bitter taste

receptor TAS2R8 in the receptor assay with high potency and

selectivity,⁷⁵eﬀectively attenuate the bitterness of coﬀee, and

display very good safety proﬁles.

RESULTS AND DISCUSSION

■

Identiﬁcation of Most Relevant TAS2Rs for Coﬀee

Bitterness. Although many bitter components of coﬀee are

known,⁷⁶⁻⁷⁸we decided not to focus our attention on a single

or a small set of bitter agonists from coﬀee. Arguably, some

individual bitter components may already have their own

bitterness blocker(s) within the coﬀee extract. Such a bitter

component may not be the main contributor of coﬀee

bitterness even if it is the most bitter compounds when

compared to other compounds of the extract in their pure

forms. Instead, we sought to identify the most relevant

receptor mediating coﬀee bitterness and to develop antagonists

of the said receptor. We hypothesized that blocking the most

relevant coﬀee TAS2R(s) would lead to a more sizable coﬀee

bitterness reduction eﬀect. We also reasoned that the

fractionation, taste testing, and receptor proﬁling of bitter

coﬀee fraction(s) could lead to the identiﬁcation of the most

relevant TAS2R(s) mediating coﬀee bitterness. Thus, a

commercial Nestle 100% Arabica dark roast instant coﬀee

sample was hot-extracted and submitted to fractionation by

reverse-phase HPLC. A total of 5 fractions were generated,

lyophilized, and evaluated in sensory for bitterness. Of the 5

fractions, fraction F4 was rated as the most bitter by sip and

spit taste testing. Subsequently, fraction F4 was screened for

the activation of TAS2Rs with cells transiently overexpressing

TAS2Rs and the promiscuous, chimeric G protein G16t44.⁷⁹

In our hands, the assays with G16t44 (Gα16 with the last 44

amino acids replaced by those from transducin) give larger

signals than other G-protein constructs. The results indicated

The identiﬁcation of potent bitter taste antagonists has been

challenging.³⁸In fact, only a few antagonists of several of the

B

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that fraction F4 mainly and strongly activated TAS2R8 and

TAS2R14. Dose−response curves of F4 activation of TAS2R8

and TAS2R14 were subsequently generated (Figure 2). It is

Figure 2. Activation of TAS2R8 and TAS2R14 by coﬀee fraction F4.

worth noting that TAS2R8 is not one of the bitter taste

receptors previously reported to be activated by caﬀeine,

namely, TAS2R7, TAS2R10, TAS2R14, TAS2R43, and

TAS2R46.⁷Interestingly, caﬀeine is believed to contribute to

less than 20% of overall coﬀee bitterness.⁷⁸Hence, based on

our data, we hypothesized that TAS2R8 antagonists may

signiﬁcantly reduce the perceived bitterness of coﬀee.

Library Screening and Background SAR. Using a

typical microtiter plate FLIPR calcium mobilization assay

developed with recombinant cells overexpressing TAS2R8 and

a promiscuous G protein,⁷⁹we screened a collection of over

200000 compounds from our corporate collection for

antagonist activity on TAS2R8. For the purpose of screening,

andrographolide (Figure 3) was used as the surrogate TAS2R8

agonist in the assay; the concentration of andrographolide used

in the screening was set to its EC80 value.

Our initial screening campaign identiﬁed about 500

compounds (0.25% hit rate) demonstrating at least 50%

inhibition of the andrographolide agonist activity at 10 μM.

Subsequent conﬁrmation and dose−response assays further

reﬁned the hit list to 9 compounds with the greatest potency

(0.005% of initial library). The most potent compound was

S5033 with an IC₅₀of 2.3 μM in the assay (Figure 4).

Following the identiﬁcation of S5033, we conducted a cluster

analysis of the initial 500 primary hits to identify S5033-related

analogues. A structure activity relationship (SAR) analysis of

the S5033-related analogues revealed that compounds

featuring the pyrazolylmethyl-3,5-dimethylisoxazole moiety,

depicted as “A”, (Figure 4) exhibit high TAS2R8 antagonist

activity relative to the other S5033 analogues. Although not

extensively investigated, any change on “A” resulted in a

signiﬁcant decrease in activity. A snapshot of the representative

analogues showcasing modiﬁcations on moiety “A” and their

impacts on the compounds’ activity is provided in the

Supporting Information. This article will focus on optimization

eﬀorts based on systematic modiﬁcations of the linker and “B”

moiety.

Figure 3. Andrographolide structure and activation of TAS2R8.

4-amine hydrochloride 6 with a set of more than 120

commercially available carboxylic acids. The carboxylic acids

were selected so that the corresponding amide coupling

products 7 would have a clogP < 4 and a molecular weight

<400. The selection also included a variety of appending

substituents in order to increase the diversity. It is worth

mentioning that the above-mentioned clogP and molecular

weight criteria were selected primarily for achieving good

solubility. Since the bitter taste receptors are located at the

upper surface of the tongue and are thus readily accessible, we

prefer compounds with rather poor bioavailability as a means

to minimizing unnecessary systemic exposure. The key

building block 6 was generated following the sequence of

reactions depicted in Scheme 1. In brief, compound 3 was

conveniently prepared from commercially available 4-(chlor-

omethyl)-3,5-dimethylisoxazole 1 and ethyl 1H-pyrazole-4-

carboxylate 2. Treatment of 3 with hydrazine in ethanol under

reﬂuxing conditions yielded compound 4, which was then

treated with acidic sodium nitrite to furnish the acyl azide 5.

Finally, Curtis’ rearrangement of 5 in tert-BuOH led to the

corresponding Boc-protected amine, which was subsequently

treated with hydrochloric acid to yield the desired amine

hydrochloride building block 6. With the amine building block

6 now in hand, the desired amide library was generated by

parallel synthesis by coupling amine 6 and the selected set of

carboxylic acids (Scheme 1) followed by HPLC puriﬁcation.

As shown in the representative set of analogues summarized

in Table 1, removing the thioethyl group of S5033 yielded the

3-pyridyl analogue 9, which was found to exhibit almost a 2-

fold increase in potency. The 2-pyridyl and 4-pyridyl analogues

8 and 10 were two- and 3-fold less active, respectively, when

compared to the 3-pyridyl (9). Replacing the thioethyl group

of S5033 with a methoxy group produced compound 11 with

almost a 2-fold improvement in potency. Moving the methoxy

group to the 4 position (13) and 5 position (12) further

improved the potency. It is worth noting that the potency of

the 2-methoxy-3-pyridyl analogue 11 was very close to that of

Optimization Based on the B moiety. To quickly

evaluate the SAR around modiﬁcations of the B moiety, a small

library of compounds with generic structure 7 was generated

by reacting 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-

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Figure 4. Structure, graphical SAR strategy, and S5033 inhibition of TAS2R8 response to andrographolide.

analogues such as indole 21, benzodioxole 23, and

benzodioxine 24 exhibited submicromolar activities. In

contrast, the TAS2R8 antagonist activity of the benzoxazole

analogue 22 was signiﬁcantly lower than the other biaryl

analogues. Phenyl analogues combining various substituents

were also synthesized. While some combinations, such as 25

which combines the 16 and 17 substituents, were not

advantageous, others, including 26 (combining 15 and 16

substituents), resulted in compounds with improved potency.

Several 5-membered aryl analogues were also synthesized but

most of them were only moderately active (e.g., 27).

Scheme 1. Synthesis of 7 and Generation of Corresponding

Library of Amides

a

While this evaluation provided compounds with good

potency in the assay (e.g., 23), we felt that such level of

potency was still insuﬃcient to allow use levels that are low

enough to warrant regulatory approval in many parts of the

globe as a bitter taste modifying ingredient. Thus, further

improvement of potency was deemed necessary. With the

valuable SAR insights obtained from these compounds, we

moved to the evaluation of the amide linker as a way of ﬁnding

other opportunities to improve the potency.

Linker Optimization. Initial linker modiﬁcations were

built around compound 14, which is the core structure of the

most potent set of compounds from early evaluations (e.g.,

14−26) and would allow us to use a range of commercially

available building blocks without the need for laborious

syntheses. The set of compounds generated as part of this

evaluation is shown in Table 2. Compounds 32, 33, and 34

were prepared in the similar manner as in Scheme 1 from the

amine 6 and the corresponding carboxylic acids. Compounds

28−31, and 35 were all synthesized in one step by treating the

amine hydrochloride building block 6 with the appropriate

electrophile in the presence of a base either at room

temperature or with heating (Schemes 2a and 2b). In some

cases, heating was performed under microwave (μW).

Compound 38 was prepared by a base- and heat-promoted

a

Reagents and conditions: (a) Cs₂CO₃, DMF, 80 °C, 16 h; (b) N₂H₄,

EtOH, reﬂux, 48 h; (c) NaNO₂, AcOH, H₂O, 0 °C to room

temperature (RT), 30 min; (d) tert-BuOH, reﬂux, 6 h; (e) MeOH,

HCl, reﬂux, 2 h.

the simple 3-pyridyl analogue 9. A similar pattern is observed

for the simple phenyl series where the activity of 14 remains

nearly unchanged with the addition of a methoxy group at the

ortho position (15) but increases to almost 3-fold when the

methoxy group is moved to either the meta position (16) or

the para position (17). Uncapping the methoxy groups to form

the corresponding ortho-phenol 18, meta-phenol 19, and para-

phenol analogues 20, resulted in a slight increase in potency for

all three analogues. We also observed that fused biaryl

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Table 1. Selected SAR Focusing on Substructure B Modiﬁcations

reaction between the benzylurea 35 and ethyl 2-chloro-2-

oxoacetate 42 (Scheme 2a). Heating the urea 43 in the

presence of a sodium hydride led to the corresponding

imidazolidone intermediate 44 that was then reacted with

benzyl chloride 45 to furnish 36 (Scheme 2a). Finally,

compound 37 was synthesized in two steps starting with the

one-pot cascade formation of unsubstituted hydantoin

intermediate 48 from acyl azide 5 and the glycine ester 47,

followed by alkylation with benzyl bromide 49 (Scheme 2c).

Screening data collected from these linker modiﬁed

compounds are summarized in Table 2. Replacing the

carboxamide bond of 14 with the sulfonamide to form

compound 28 resulted in a loss of activity. Inserting a

methylene group between the amide nitrogen and the phenyl

ring of 14 to form the isoindolidone 29 induced an even higher

drop in potency. However, inserting a carboxyl rather than a

methylene group to form the isoindolidione 30 resulted in a

signiﬁcant boost in potency, which was about 8-fold relative to

amide 14. Compounds with improved potencies were also

obtained when the phenyl analogue 14 was elongated by

insertion of methylene, ethylene, or propylene linkers between

the amide carbonyl and the phenyl group, resulting in

compounds 32, 33, and 34, respectively. Within this series of

compounds, the two carbon analogue 33 was found to provide

greater activity. Thus, we decided to evaluate further

modiﬁcation of the linker without signiﬁcant alteration of the

length with regard to compound 33. With that in mind, the

urea 35 and the corresponding imidazolidinones 36, 37, and

38 were synthesized. Although the urea 35, the imidazolidi-

none 36, and the imidazolidinetrione 38 were all less active

than 33, we were pleased to ﬁnd that the hydantoin 37

exhibited a 2-fold potency improvement relative to 33. Thus,

from this set of analogues targeting optimization of the linker,

the isoindolidinone 30 and the hydantoin 37 emerged as the

two most potent analogues. Both compounds also have low

molecular weights, as desired at this stage of optimization: MW

= 322 for 30 and MW = 365 for 37.

In order to diﬀerentiate the two most potent compounds

and select the most promising lead for further optimization,

solubility and stability data for 30 and 37 were collected

(Table 3). The decision to collect high-temperature stability

data for these compounds, rather than simple shelf-life stability

data, was based on the need for the product candidates to be

stable for applications in foods, beverages, and pharmaceuticals

E

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analogues was generated. Selected members of the library are

shown in Table 4. For the synthesis of the desired analogues,

the key building block 48 was scaled up following the

procedure depicted in Schemes 2a−2c and then alkylated

under basic conditions using the appropriate building blocks

(Scheme 3). In cases where the substituent R was an ester, the

products were hydrolyzed to furnish the corresponding

phenols (e.g., 55 and 56) or carboxylic acids (e.g., 58).

Alternatively, 55 and 56 were prepared as in Scheme 5 with or

without protection of the phenol; however, using a protecting

group clearly improved both the yield and impurity proﬁles.

The latter carboxylic acid 58 was further modiﬁed by amide

coupling to yield analogues such as 59.

Table 2. Selected SAR Focusing on Linker Modiﬁcations

As far as TAS2R8 antagonist activity is concerned, a benzyl

group with small substituents such as ﬂuorine, methyl,

methoxy, cyano, and hydroxyl was, in general, well tolerated

(51 − 56, Table 4); however, some (e.g., 54) were less active

than the lead compound 37. However, the hydroxyl group

appeared to be a substituent of choice, exhibiting double digit

nanomolar IC₅₀values regardless of its position on the benzyl

ring: 55, 56, and S6821 (Table 4 and Figure 5). The

introduction of carboxylic acid substituents (e.g., 58) resulted

in a drop of activity, whereas the corresponding esters (e.g.,

57) and small amides such as 59 improved the activity slightly

when compared to the lead compound 37; however, it is still

less potent than the corresponding hydroxyl analogues.

Extending the hydroxyl group to form hydroxyl-methyl

analogues (e.g., 60) resulted in a moderate drop of activity.

Alternatively, the phenyl ring of the benzyl group was also

replaced either with cycloalkyls (e.g., 62), heterocycloalkyls

(e.g., 63), or heteroaryls (e.g., 61, 64, and 65). While the

cyclohexylmethyl 62 retained the potency, the tetrahydrofur-

ylmethyl 63 was about 5-fold less potent than the lead

compound 37. On the other hand, the pyridylmethyl 61, the

pyrrazole 64, and the oxazolylmethyl 65 were all reasonably

well tolerated.

With this set of highly potent analogues identiﬁed, it was

necessary to assess them against the desired characteristics of

potential product candidates. For optimal use level and safety

margin, the compounds of interest were required to display

high in vitro TAS2R8 antagonist activity with at least a two-

digit nanomolar IC₅₀obtained from averaging several sets of

data from diﬀerent screening days. To allow for a convenient

formulation in various media, the solubility at pH 7.1 was

required to be at least 50 μM and 1000 times greater than the

IC₅₀. The desired compounds were also required to exhibit less

than 5% degradation under most standard processing

conditions, which include retorting (up to 60 min at 124

°C) and ultra-high-temperature processing (UHT, 30 s at 140

°C). Ultimately, the compound should exhibit an excellent

safety proﬁle. With this set of characteristics in mind, a

sequential proﬁling of our most potent compounds, including

compounds 51, 53, and S6821 (Table 4), was undertaken.

Like 51 and 53, some compounds exhibiting the desired

potency had solubility far less than the desired 1000 times IC₅₀

(Table 4). Thus, these compounds were not further

considered. Since compound S6821 satisﬁed both potency

and solubility requirements, its stability was assessed under a

variety of storage and processing conditions. The results are

summarized in Table 5. In fact, while S6821 was very stable

under most of the conditions tested, considerable degradation

was observed when a solution of S6821 at pH 7.1 was heated

either at 100 °C for 24 h or for 60 min at 124 °C. Degradation

that often require harsh processing conditions. The results

were clearly in favor of 37, which showed signiﬁcantly better

stability, with 60% of the parent compound remaining after 24

h at 100 °C, pH 7.1 and >99% remaining after 24 h at 100 °C,

pH 2.8. Under the same conditions, 30 was signiﬁcantly less

stable, where 5% was remaining after 24 h at 100 °C and pH

7.1 and 3% remaining after 24 h at 100 °C and pH 2.8. In

addition, with 12 μM solubility in a pH 7.1 buﬀer, the

isoindolidione 30 was about ﬁve times less soluble than the

hydantoin 37, which exhibited a 70 μM solubility under the

same conditions (Table 3). In light of these results, the

hydantoin 37 was selected as the lead compound for further

optimization.

SAR Reﬁnement of the Lead Compound 37. With 37

now identiﬁed as the lead compound for further SAR

reﬁnement, we decided to focus further modiﬁcations ﬁrst on

the decoration of the benzyl group of 37 with small

substituents (e.g., 51−60) and the replacement of the benzene

ring with a variety of ﬁve- and six-membered alkyl and aryl

rings (e.g., 61 − 65). Thus, a small diverse library of about 50

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a

Scheme 2a. Synthesis of Compounds 28, 29, 31, 35, 36, and 38

a

Reagents and conditions: (a) Et₃N, ACN, μW, 135 °C, 5 min; (b) NaH, DMF, μW, 150 °C, 10 min; (c) Et₃N, ACN, reﬂux, 3 h; (d) NaH, ACN,

μW, 120 °C, 10 min.

a

solution (HBSS) was suﬃcient for cells to recover from agonist

stimulation with andrographolide (data not shown). Cells were

then repeatedly stimulated with either 100 μM andrographo-

lide or with 100 μM andrographolide and 1 μM S6821 (Figure

6A and B). As expected, 1 μM S6821 signiﬁcantly blocked the

response of all andrographolide responding cells. Furthermore,

a complete recovery of the agonist activity was observed

following a brief 5 min washout, thus indicating that the

binding of S6821 to TAS2R8 is completely reversible. The

same results were also observed using 100 μM chloramphe-

nicol and 1 μM S6821 (Figure 7).

Further mechanistic investigations of the S6821 interaction

with TAS2R8 were also conducted. We performed a dose−

response analysis of TAS2R8 with increasing concentrations of

the agonist chloramphenicol and ﬁxed concentrations of

S6821. Dose−response curves of TAS2R8 with chloramphe-

nicol alone resulted in an EC₅₀of 41 μM. Increasing the

concentrations of S6821, successively, resulted in successive

rightward shifts in the chloramphenicol dose−response curves.

At S6821 concentrations above 30 nM, the highest

chloramphenicol concentration (2 mM) could not be

Scheme 2b. Synthesis of Compound 30

a

Reagents and conditions: (a) Cs₂CO₃, DMF, reﬂux, 80 °C, 3 h.

of S6821 was also observed in strongly basic solutions, e.g.,

∼20% remaining in pH 9.3 after 24 h at RT. In all these cases,

the degradative pathway was found to be the hydrolytic

opening of the hydantoin ring of S6821 to form 66. The latter

reaction is, however, reversible, with 66 rapidly cyclizing back

to S6821 below neutral pH (Scheme 4).

To see if the antagonist activity of S6821 on TAS2R8 was

reversible, we conducted a ratiometric calcium imaging

experiment using a multichannel perfusion system, so the

compounds could be applied reversibly or “washed out”

(Figure 6). In a preliminary set of experiments, we ﬁrst

determined that a 5 min washout time by Hank’s balanced salt

a

Scheme 2c. Synthesis of Compound 37

a

Reagents and conditions: (a) toluene, reﬂux, 1 h; (b) Et₃N, toluene, reﬂux, 16 h; (c) Cs₂CO₃, DMF, μW, 85 °C, 20 min.

G

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Table 3. Solubility and Stability of Compounds 30 and 37

*

**

After 24 h at pH 7.1. After 24 h at 100 °C.

concentrations above 1−2 mM the assay response begins to

decrease. At these high concentrations, either (1) chloram-

phenicol begins to inhibit TAS2R8 or (2) some other

nonspeciﬁc inhibitory eﬀect reduces the ﬂuorescent signal.

While this observation could suggest an insurmountable

noncompetitive mechanism, the dose−response curve ﬁts the

convergence to a similar value of Emax, the equidistant dextral

right shifts in the chloramphenicol dose−responses, and the

Schild regression analysis point to a surmountable competitive

mechanism of action (Figure 8).

Table 4. Selected Compounds for Lead Reﬁnement Based

on Right Hand Side Modiﬁcations

S6821 antagonist proﬁle was also evaluated with transiently

transfected cells for relative selectivity against a panel of 16

TAS2Rs, and the results are shown in Figure 9; the list of

agonists used for the panel screening and the corresponding

TAS2R targets are provided in Table 6. It was determined

from these studies that at concentrations up to 25 μM, S6821

exhibits high TAS2R8 selectivity across the entire panel of the

16 TAS2Rs screened (Figure 9 and Table 6). TAS2R8 activity

was inhibited by about 90% in the presence of 25 μM S6821,

and only TAS2R39 was also poorly antagonized. However,

although the eﬀect of 25 μM S6821 on other TAS2Rs was

found to be poor, it would not be surprising to observe a more

promiscuous behavior at higher concentrations. In fact, despite

the low sequence similarity between TAS2Rs, bitter antago-

nists reported thus far have been shown to be promiscuous

toward a range of TAS2Rs.^67,69

While the relatively high TAS2R8 selectivity of S6821 could

be viewed as a limitation to its antagonist eﬀect on a broader

range of bitter tastants, we hypothesized that this high

selectivity could also be a good indication of a limited safety

liability for S6821. The potential toxicity of S6821 was initially

assessed on the basis of structural alerts and on the basis of

safety information available for its component fragments.⁸⁰⁻⁸²

Since the latter assessment did not reveal any structural alerts,

S6821 was not expected to be carcinogenic or mutagenic. In

light of these results and given its high potency, selectivity, and

solubility as well as its good stability proﬁles across a number

of storage and processing conditions, S6821 was selected for

developmental proﬁling. S6821 was not found to be mutagenic

or clastogenic in vitro and did not induce micronuclei in bone

marrow polychromatic erythrocytes in vivo.⁷⁵Phase I

metabolites M397A, M397B, and M397C resulting from

monohydroxylation at various positions of the phenol ring

were identiﬁed during an in vitro metabolism study of S6821

using rat and human liver microsomes (Scheme 5 and ref 75).

In rat pharmacokinetic (PK) studies, the majority of S6821

was found to rapidly convert to the corresponding sulfate

M461 and glucuronide M557 (Scheme 6). Of the phase I

*

After 24 h at pH 7.1.

produced; a signal equivalent to the maximum receptor

response and higher concentrations of chloramphenicol

interfered with the assay. In fact, we observed that at

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a

Scheme 3. Synthesis of Selected Compounds 50−65

a

Reagents and conditions: (a) base, solvent, μW, or conventional heating; (b) condition (a), NaOH, HCl; (c) DIEA, HOBt, EDCI.

Figure 5. S6821 potently inhibits TAS2R response to andrographolide.

Table 5. Selected Data for S6821

stability test conditions

a

compound

TAS2R8 IC₅₀(μM)

solubility (μM)

solubility/IC₅₀

11619

temperature

pH

time

24 h

60 min

30 s

% remaining

S6821

0.02

244

RT

100 °C

124 °C

140 °C

2.8

5.0

7.1

9.3

2.8

5.0

7.1

6.0

7.1

6.5

>99%

∼20%

>99%

∼55%

>99%

∼89%

>99%

a

After 24 h at pH 7.1.

Scheme 4. Temperature and pH-Dependent Stability of S6821

metabolites detected in vitro, only M397C was detected in

vivo.⁷⁵

In vivo 28-day dose-range-ﬁnding toxicity studies of S6821

determined the maximum tolerated dose (MTD) to be above

100 mg/kg/day (the highest dose tested) for CD rats, and no

S6821 treatment-related adverse events were observed for the

90-day repeat-dose toxicity study.⁷⁵Thus, the no-observed

adverse-eﬀect level (NOAEL) for S6821 was 100 mg/kg of

body weight (bw)/day, which was the highest dose tested.

S6821 also demonstrated a NOAEL of 1000 mg/kg of bw/day

(the highest dose tested) for both maternal toxicity and

embryo/fetal development following oral administration to

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Figure 7. S6821 reversibly blocks chloramphenicol activation of

TAS2R8-expressing cells. (A) Representative experiment showing

average calcium response of all chloramphenicol responding cells

during repeated 100 μM chloramphenicol stimulations either with or

without inhibitor S6821 (1 μM). (B) Distribution of peak amplitude

of all cells during 3 consecutive chloramphenicol applications with

initial chloramphenicol application (control, black bars), in the

presence of S6821 (Gray Bars) and following wash-out (Recovery,

White Bars). Average peak ( SEM) responses for the three

applications are showed in the inset. Note the signiﬁcant block of

chloramphenicol by S6821 (P < 0.0001, n = 171 cells, two sample t

test). No reduction was found between the ﬁrst and the third

chloramphenicol responses, suggesting complete recovery/washout of

S6821. Data are from 171 total cells from 3 separate imaging

experiments.

Figure 6. S6821 reversibly blocks andrographolide activation of

TAS2R8-expressing cells. (A) Representative experiment showing

average calcium response of all andrographolide responding cells

during repeated 100 μM andrographolide stimulations either with or

without inhibitor S6821 (1 μM). (B) Distribution of peak amplitude

of all cells during 3 consecutive andrographolide applications with

initial andrographolide application (control, black bars), in the

presence of S6821 (gray bars) and following wash-out (recovery,

white bars). Average peak ( SEM) responses for the three

applications are showed in the inset. Note the signiﬁcant block of

andrographolide by S6821 (P < 0.0001, n = 136 cells, two sample t

test). No signiﬁcant diﬀerence was found between the ﬁrst and the

third andrographolide responses (P = 0.24, two sample t test),

suggesting complete recovery/washout of S6821. Data are from 136

total cells from 3 separate imaging experiments.

pregnant rats during gestation.⁷⁵The detailed ADME-PK,

genotoxicity, and toxicity studies conducted as part of the

S6821 development campaign have been previously re-

ported.⁷⁵Details of the syntheses of M397A, M397B, and

M397C (Scheme 5) as well as M461 and M557 are described

herein for the ﬁrst time.

Since some TAS2R antagonists have been reported to also

be agonists of other TAS2Rs,⁵we decided to check if S6821

could be activating other TAS2R(s) in our cell-based assays.

Thus, the S6821 TAS2Rs agonist proﬁle was evaluated on

transiently transfected cells. The results showed stimulation of

TAS2R1 and TAS2R14 at 25 μM S6821. Subsequent dose−

response assays on stable cells lines revealed EC50 values of

150 μM and 40 μM against TAS2R1 and TAS2R14,

respectively (Figure 10). S6821 was ultimately sensory-

evaluated for intrinsic bitterness and was not signiﬁcantly

bitter up to 150 μM. On a 15-point bitterness scale, 150 μM

bitterness through the activation of other TAS2Rs (e.g.,

TAS2R14).

To assess its bitterness blocking eﬃcacy, S6821 was

evaluated in taste tests for the reduction of bitterness in Alta

Rica instant coﬀee. A sample containing 22.5 g of Alta Rica

100% Arabica instant coﬀee in 750 mL of water and 250 mL of

whole milk was ﬁrst tasted alone and deemed strongly to very

strongly bitter by 13 out of 17 panelists. At the same time 3 out

of 17 panelists rated the sample as moderately bitter and only 1

out of the 17 panelists found it to be weakly bitter. When the

coﬀee sample was compared to a coﬀee sample containing 5

μM S6821, more than 78% of responses had the sample

containing S6821 as being less bitter, with a p-value <0.001

(Figure 11). These results are likely inﬂuenced by variations in

TAS2Rs genetic coding sequences between panelists. In fact,

interindividual genetic polymorphisms have been found to

have a signiﬁcant eﬀect on human sensitivity to and/or liking

of coﬀee.^50,52,54,55

S6821 scored 2.8

0.6 on average, and panelists found no

signiﬁcant diﬀerence in bitterness among low sodium buﬀer

solution (LSB), LSB + 100 μM S6821, and LSB + 150 μM

S6821 samples (p-value >0.05), as shown in Table 7. Hence, at

low concentrations, S6821 was expected to eﬀectively block

TAS2R8-mediated bitterness without signiﬁcantly adding

SAR Targeting Potent and Soluble Analogues with

Broader Stability Proﬁle. Encouraged by the very positive

safety data obtained with S6821, we decided to undertake

additional eﬀorts aimed at identifying an analogue with an

improved hydrolytic stability proﬁle as a backup for S6821.

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Figure 8. S6821 inhibits chloramphenicol activation of TAS2R8 in a competitive manner. (A) TAS2R8-expressing cells were treated with

increasing concentrations of chloramphenicol in the absence or presence of 3 nM to 3000 nM S6821, and receptor response was measured in the

cell-based assay. Data were calculated as the mean s.d. of assay duplicates. Similar results were obtained in 3 independent experiments. (B) Schild

regression analysis from dose−response curves in panel (A).

Figure 9. TAS2Rs panel screening of S6821 indicates a relatively high selectivity for TAS2R8; refer to Table 6 for details on agonists used.

The strategy was to add substituents to the methylene moiety

of the hydantoin ring in order to hinder the access of water and

other nucleophiles to the vulnerable carbonyl site and thus

slow the hydrolysis of the hydantoin ring. With that in mind,

compounds 71−76 were synthesized following the reaction

sequence shown in Scheme 5.

Compounds generated were once again screened on stable

cells expressing TAS2R8 in the presence of andrographolide as

agonist. To our delight, most of the generated compounds

retained double digit nanomolar inhibitory activity (Figure 12

and Table 8). In addition, compounds 71−74 and S7958 still

met our predeﬁned solubility criteria of at least 50 μM and

1000 times IC₅₀. However, the stability of compounds 73 and

74 was unexpectedly low. Arguably, in an aqueous solution, the

hydroxymethyl group present in the latter compounds may

coordinate with a water molecule to help drive the hydrolysis

of the adjacent carbonyl moiety. Gratifyingly, S7958 suﬀered

less than 1% hydrolysis when heated with pH 7.1 for 24 h at

100 °C and for 60 min at 124 °C (Table 8), which was a

signiﬁcant improvement over S6821 under the same

conditions (Table 5 and 8). The impressive hydrolytic stability

of S7958 was also evidenced in a strong basic pH, with S7958

exhibiting no noticeable degradation even after 24 h with pH

9.3 at RT. S7958 was also evaluated for antagonist activity on

other TAS2Rs and was found to exhibit an inhibition proﬁle

that was very similar to that of S6821 (Table 8).

Thus, given the excellent stability, solubility, and TAS2R8

antagonist activity of S7958 and the expectation that it may

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a

Table 6. Panel of TAS2Rs and Corresponding Agonists Used to Access the Putative S6821 and S7958 Antagonist Eﬀect

a

Relative activity remaining: <25% (green), 25−75% (yellow), >75% (red)

levels of intake by the Joint FAO/WHO Expert Committee on

Food Additives and were assigned JECFA Nos. 2161 and

2162,⁸⁵respectively. In addition, the Panel on Food Contact

Materials, Enzymes, Flavorings and Processing Aids of The

European Food Safety Authority (EFSA) has determined that

S6821 (FL-no, 16.127) is not expected to be of a safety

concern when used at levels up to those speciﬁed in diﬀerent

foods.⁸⁶Regulatory jurisdictions in countries such as Japan,

Korea, and Mexico (794 DO 5.9.2013) have also approved the

use of S6821.

Scheme 5. Synthesis of 71−76, S6821, S7958, M397A,

a

M397B, and M397C

CONCLUSION

■

We have disclosed a new class of 3-(pyrazol-4-yl)-imidazoli-

dine-2,4-diones as potent, selective, and eﬃcacious TAS2R8

antagonists. These include 3-(1-((3,5-dimethylisoxazol-4-yl)-

methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)imidazolidine-

2,4-dione (S6821) and 3-(1-((3,5-dimethylisoxazol-4-yl)-

methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5,5-dimethyli-

midazolidine-2,4-dione (S7958), which have demonstrated the

ability to signiﬁcantly reduce the bitterness elicited by coﬀee.

We demonstrated that the bitter fractions from roasted coﬀee

extracts strongly activate TAS2R8. Thus, it is expected that

S6821 and S7958 will also be eﬀective in blocking TAS2R8-

mediated bitterness associated with other products such as

APIs, excipients, foods, beverages, and nutraceuticals. S6821

and S7958 have been tested in a panel of toxicity studies and

were determined by US, Europe, and other national and global

regulatory expert panels to be safe under intended conditions

of use as bitter antagonists. Driven by this successful story, we

are currently pursuing the identiﬁcation and optimization of

antagonists for other TAS2Rs.

a

Reagents and conditions: (a) imidazole or DIEA, DCM; (b)

NaBH(OAc)₃, Et₃N, DCE; (c) toluene, reﬂux; (d) aq. HCl.

also exhibit a safety proﬁle similar to that of S6821, we decided

to move S7958 into development as a S6821 backup.

Subsequent in vitro and in vivo safety evaluations conducted

as part of S7958 development have also been reported.⁷⁵As

expected, the ADME-PK, genotoxicity, and toxicity proﬁle of

S7958 were all in line with the positive data obtained for

S6821, as summarized above. In line with its potency, S7958

also showed a signiﬁcant block of coﬀee bitterness in taste test

(Table 9).

Regulatory Status and Evolving Product Application

of S6821 and S7958. In light of these safety and eﬃcacy

data, S6821 and S7958 were determined to be “Generally

Recognized As Safe” by the Flavor and Extract Manufacturers

Association (FEMA) expert panel and were assigned FEMA

GRAS Numbers 4725 and 4726,^83,84respectively. S6821 and

S7958 have also been determined to be safe at the current

EXPERIMENTAL SECTION

■

Biology. Ratiometric Calcium Imaging Methods. Recombinant

cells stably expressing TAS2R8 were grown overnight on Matrgel-

coated glass coverslips. Prior to imaging, cells were incubated with 3

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a

Scheme 6. Synthesis of M461 Sodium Salt and M557

a

Reagents and conditions: (a) silver triﬂate, DCM, overnight; (b) NaOH, MeOH, overnight; (c) HCl, MeOH/H₂O, (d) pyridine SO₃(PySO₃),

DCM, reﬂux, 2 h; (e) NaHCO₃.

Figure 10. Dose−response evaluation of S6821 against TAS2R1 and

TAS2R14.

Figure 11. Number of responses selecting each sample as more bitter.

N = 51 responses (17 panelists × 3 replicates).

Table 7. Perceived Bitterness Intensity Scores of S6821 on a

Scale of 1−15

a

standard

deviation

Tukey’s (5%)

lettering

treatment

average

LSB + 50 μM S6821

LSB

LSB + 100 μM S6821

LSB + 150 μM S6821

1.0

1.6

1.9

2.8

1.0

2.1

1.8

3.1

A

Ab

B

a

N = 30 responses (15 panelists × 2 replicates). Tukey’s value = 1.155

(α = 0.05).

μg/mL FURA-2AM for 1 h at RT in HBSS. For imaging, cells were

mounted on a ﬂuorescent microscope equipped with a multichannel

perfusion system and continuously perfused with HBSS and TAS2R8

agonist andrographolide (100 μM) or chloramphenicol (100 μM).

For S6821 inhibition experiments, 1 μM of the inhibitor was applied

for 60 s prior to and during agonist application. All compounds were

prepared from DMSO stock, and the ﬁnal DMSO concentrations did

not exceed 0.1%. During acquisition, all cells in the ﬁeld were

autoselected by the software as regions of interest (ROIs) for analysis.

Measurement of intracellular calcium levels were acquired at 1 Hz

using 100 ms of excitation at 340 and 380 nm, and the emissions were

monitored by a charge-coupled device (CCD) camera. The average

response of all ROIs in the ﬁeld and the peak responses for each cell

were measured during each application. For S6821 recovery

experiments, a 5 min wash-out period was determined suﬃcient for

cells to fully recover from repetitive agonist stimulation, and only cells

Figure 12. S7958 inhibition of TAS2R8 response to andrographolide.

with a strong initial TAS2R8 agonist responses (ΔF/F > 0.5) were

selected for analysis.

TAS2R8 Stable Cell Line. Recombinant cells stably expressing the

G protein chimera G16t44 and TAS2R8 were generated by

transfection using standard lipofection techniques, and individual

clones were isolated by limiting dilution. Clones were initially

screened by responses to andrographolide in calcium imaging assays

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Table 8. Selected Data for S6821 Analogues Targeting Stability Improvement

a

b

After 24 h at pH 7.1. After 24 h at 100 °C.

Sensory Methods and Data Reporting. The taste test and

protocol used for evaluation by human panelists were reviewed and

conducted according to guidance from external toxicology and

regulatory experts in the ﬁeld. Sensory evaluations were conducted

after approval by the Firmenich Internal Review Board, and taste

samples were only presented to panelists after written informed

consent. The data and protocol packages associated with the title

compounds have been reviewed by qualiﬁed regulatory authorities.

Overview of Taste Test Methods. Two diﬀerent types of sensory

tests were used to evaluate S6821 in either the inherent bitterness or

blocking bitterness of coﬀee. The conducted taste tests include the

following: (1) A sensory scaling test was performed using a 15-point

bitterness scale in order to evaluate the reduction of bitterness

intensity of S6821 alone. (2) A paired comparison test (2-alternative

forced choice diﬀerence tests) was used to demonstrate a signiﬁcant

reduction in bitterness for coﬀee with S6821 added as compared to

coﬀee alone.

Stimuli. Samples for the scaling evaluation were performed with

solutions of S6821 at various concentrations (0, 100, and 150 μM).

For these studies, S6821 was prepared as a concentrated stock

solution in ethanol to ensure dissolution of the compound. Samples

without compound within the same test were balanced with an

equivalent concentration of ethanol. The ﬁnal concentration of

ethanol was 0.1%. Solutions were prepared in low sodium phosphate

buﬀer (LSB; pH 7.1). LSB consists of 0.3 mM KCl, 0.5 mM

Na2HPO4, and 0.175 mM KH2PO4.

Table 9. Number of Times Selected As More Bitter by

Panelists

a

taste test with

samples

S6821

S7958

1.5× Alta Rica Course (ARC) +

milk

40

33

1.5× ARC + milk + 5 μM S6821

1.5× ARC + milk + 5 μM S7958

total responses

11

18

51

1.5× ARC + milk selected (p-

value)

<0.001

0.049

a

N = 51 responses (17 panelists × 3 reps).

using ﬂuo4-loaded cells on a ﬂuorescent microscope. Clones showing

the best response were characterized further on FLIPR.

FLIPR Assay. Recombinant cells stably expressing the TAS2R8

receptor were plated in a 384-well plate 24 h prior to the assay. On

the day of the assay, the cells were incubated with ﬂuo-4 dye in

phosphate-buﬀered saline (PBS) for 60 min followed by 3 washes

with PBS. Cells were subsequently resuspended in PBS and allowed to

sit in the dark at room temperature for 15 min immediately prior to

the assay. Compounds were diluted in PBS/DMSO at 2× the ﬁnal

assay concentration. The ﬁnal DMSO concentration in the assay was

0.5%. For antagonist assays, the agonist and antagonist were premixed

and added simultaneously. At the beginning of the assay, the baseline

ﬂuorescence was read on a ﬂuorescence imaging plate reader (FLIPR,

Molecular Devices) for 10 s prior to the addition of compounds. An

equal volume of the compound was added to the cell plate, and

changes in ﬂuorescence were measured for an additional 100 s.

Samples for the 2-alternative forced choice test (2-AFC) were

performed with coﬀee samples prepared as follows: Alta Rica 100%

Arabica instant coﬀee was prepared with 22.5 g of dry instant coﬀee in

750 mL of boiling water, to which 250 mL of whole milk was added

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(control sample). The test sample used this same coﬀee preparation,

to which the S6821 stock solution (see above) was added to make a

ﬁnal concentration of 5 μM S6821.

Subjects. External trained panelists were used for both types of

tests. Panelists have signiﬁcant experience in evaluating bitterness

intensity in various food products. Subjects were instructed not to eat

or drink anything (except water) for at least 1 h prior to any test.

Panelists were trained to use scaling procedures and were previously

familiar with the sensory test methods used. The exact number of

panelists used is reported with each test.

Taste Test Procedures. Panelists rinsed their mouths with water

prior to starting any test. In scaling tests, panelists rated samples for

bitterness. Samples were presented to panelists monadically, in a

randomized, counterbalanced order. Panelists rinsed their mouths

with water and had up to 1 min of delay to clear the mouth of any

tastes after each evaluation. Two replicates of each test were

performed. All samples were evaluated then expectorated (i.e., “sip

and spit”).

In the 2-AFC tests, panelists tasted pairs of solutions and indicated

which sample was perceived to be more bitter. Pairs were provided in

a randomized counterbalanced order and panelists completed three

replicate tests. Between pairs, panelists rinsed with water and had up

to a 1 min delay to clear the mouth of any tastes. Prior to the 2-AFC

tests, the subjects were given a sample of the solution or product

alone, and asked to describe its bitterness intensity using one of the

following descriptors: none, barely detectable, weak, moderate, strong,

and very strong.

zole-4-carboxylate 3 (86 g, 345 mmol) and hydrazine (110 g, 3454

mmol) were stirred in EtOH (1 L) at reﬂux for 48 h. LC-MS

indicated that the reaction was complete at this point. The solution

was concentrated under a vacuum, and the solid product was

recrystallized from ethanol to aﬀord the desired compound 4 (77 g,

328 mmol, 96%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ

2.14 (s, 3H), 2.41 (s, 3H), 4.32 (br s, 2H), 5.17 (s, 2H), 7.83 (d, J =

0.8 Hz, 1H), 8.18 (d, J = 0.8 Hz, 1H), 9.31 (br s, 1H). LC-MS (ESI):

m/z 236 [M + H]⁺.

1-((3,5-Dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl

Azide (5). Note that the product 5 must be handled with care. Although

we did not experienced any issues with the acyl azide product 5, such azide

materials are known to decompose violently when friction and/or shock

are applied.

Sodium nitrite (14 g, 202 mmol) in H₂O (700 mL) was added

dropwise over 30 min to a solution of 1-((3,5-dimethylisoxazol-4-

yl)methyl)-1H-pyrazole-4-carbohydrazide 4 (31.6 g, 134 mmol) in

10% aqueous acetic acid (2 L) at 0 °C (ice water bath). The reaction

mixture became cloudy with a white precipitate during the addition of

sodium nitrite. After the addition, the ice bath was removed, and the

reaction mixture was stirred for an additional 30 min and was then

allowed to warm to room temperature. The mixture was extracted

with ethyl acetate (3×, 300 mL). The combined organic extract was

washed successively with aqueous saturated sodium carbonate, water,

and brine. The organic phase was dried over sodium sulfate, ﬁltered,

and concentrated on a rotovap. The residue was recrystallized from

ethanol to aﬀord compound 5 (31 g, 126 mmol, 93%) as a white solid.

¹H NMR (DMSO-d₆, 400 MHz): δ 2.15 (s, 3H), 2.42 (s, 3H), 5.23

Sensory Data Reporting. The statistical signiﬁcance for scaling

tests was calculated using a two-way ANOVA with interaction with α

= 0.05. The statistical signiﬁcance for the 2-AFC tests was calculated

using binomial probability tables for α = 0.05.

(s, 2H), 7.95 (d, J = 0.8 Hz, 1H), 8.57 (d, J = 0.8 Hz, 1H). LC-MS

(ESI): m/z 247 [M + H]⁺.

1-((3,5-Dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-amine Hy-

drogen Chloride (6). To a solution of 5 (1.00 g, 4.06 mmol) in

anhydrous toluene were added 4 Å molecular sieves (MS) (2 g), and

the mixture was reﬂuxed for 2 h. After cooling to room temperature,

tert-BuOH (50 mL) was added, and the mixture was heated again at

reﬂux for 6 h. The reaction mixture was cooled to room temperature,

ﬁltered over a pad of celite, and the solvent was removed under a

vacuum; the residue was triturated in hot Et₂O. The impurity that

precipitated was ﬁltered oﬀ, and the ethereal ﬁltrate was dried on the

rotovap to furnish tert-butyl (1-((3,5-dimethylisoxazol-4-yl)methyl)-

1H-pyrazol-4-yl)carbamate (LC-MS (ESI): m/z 293 [M + H]⁺). The

latter carbamate intermediate was dissolved without further

puriﬁcation in anhydrous methanol (50 mL) and treated with HCl

in Et₂O (10 equiv). The mixture was reﬂuxed for 2 h and

concentrated under a vacuum to give a residue that was recrystallized

from MeOH/Et₂O to aﬀord the desired amine hydrochloric acid 6 as

Chemistry. General Procedures. The synthesis and character-

ization of 8, 19, and 23 (Table 1); 30, 32 and 37 (Table 2); S6821

and S7958 (Table 8); M397A, M397B, and M397C (Scheme 5); and

M461 sodium salt and M557 (Scheme 6) are described below. The

synthesis of all other compounds and their intermediates and

corresponding characterization data are provided in the Supporting

Information.

Materials and Methods. Unless otherwise noted, all compounds

obtained from commercial sources were used without further

puriﬁcation. All tested compounds have a purity >95%, per one of

the HPLC methods provided in the Supporting Information. The

provided ¹H NMR, ¹³C NMR, mass spectrometry (MS), and

elemental analysis (EA) data were in all cases consistent with the

proposed structures. For NMR, the chemical shifts (δ) are in ppm,

and spectra were referenced using the residual solvent peak; coupling

constants (J) are in hertz. The following abbreviations were used for

common solvents: deuterated dimethyl sulfoxide (DMSO-d₆) and

1

a whitish solid (0.70 g, 3.06 mmol, 75% over two steps). H NMR

(DMSO-d₆, 400 MHz): δ 2.14 (s, 3H), 2.41 (s, 3H), 5.18 (s, 2H),

7.54 (d, J = 0.8 Hz, 1H), 8.03 (d, J = 0.8 Hz, 1H), 10.12 (br s, 3H).

LC-MS (ESI): m/z 193 [M_{free base}+ H]⁺. HPLC purity >95%.

General Procedure A: N-(1-((3,5-Dimethylisoxazol-4-yl)methyl)-

1H-pyrazol-4-yl)picolinamide (8). The amine hydrochloride 6 (400

mg, 2.1 mmol), picolinic acid (256 mg, 2.1 mmol), and HOBt (388

mg, 2.50 mmol) were mixed in dichloromethane (7 mL). The

reaction mixture was treated with triethylamine (670 mL, 4.8 mmol)

and then stirred for 15 min at room temperature under a nitrogen

atmosphere. Then, EDC (598 mg, 3.1 mmol) was added, and the

reaction mixture was continued to be stirred for an additional 4 h.

The reaction mixture was then diluted with dichloromethane (5 mL),

washed with a saturated NaHCO₃solution (2 × 5 mL), and washed

with a saturated NaCl solution (5 mL). The organic layer was

collected, dried over sodium sulfate, ﬁltered, and evaporated on the

rotovap. The crude product was puriﬁed by reverse phase HPLC

(H₂O/acetonitrile 10 to 90%) to aﬀord the title compound 8 as a

1

deuterated chloroform (CDCl₃). The description of H NMR peaks

was performed using the following conventional abbreviations: singlet

(s), doublet (d), triplet (t), quartet (q), multiplet (m), doublet of

doublets (dd), doublet of triplets (dt), doublet of doublets of doublets

(ddd), and broad (br). Mass spectra (m/z) were recorded using

electrospray ionization (ESI). Elemental Analyses were performed

either by NuMega Resonance Laboratories or Exova Group plc.

Ethyl 1-((3,5-Dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-car-

boxylate (3). Ethyl 1H-pyrazole-4-carboxylate 1 (21 g, 147 mmol),

4-chloromethyl-3,5-dimethylisoxazole 2 (25 g, 172 mmol), and

cesium carbonate (48 g, 147 mmol) were stirred in DMF (250

mL) under N₂at 80 °C for 16 h. The reaction mixture was cooled and

ﬁltered to remove inorganic salt. The liquid was diluted with 0.1 N

HCl (200 mL) and then diluted with water (1 L) in which a white

precipitated formed. The solid was collected by ﬁltration, washed with

water, and dried under a vacuum to aﬀord the title compound 3 (35 g,

141 mmol, 97%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz): δ

1.25 (t, J = 7.2 Hz, 3H), 2.14 (s, 3H), 2.41 (s, 3H), 4.20 (q, J = 7.2

Hz, 2H), 5.20 (s, 2H), 7.86 (d, J = 0.8 Hz, 1H), 8.43 (d, J = 0.8 Hz,

1H). LC-MS (ESI): m/z 250 [M + H]⁺.

1

white solid (374.6 mg, 1.26 mmol, 60%). H NMR (CDCl₃, 400

MHz): δ 2.21 (s, 3H), 2.44 (s, 3H), 5.05 (s, 2H), 7.49−7.47 (m, 1H),

7.60 (d, J = 0.8 Hz, 1H), 7.93−7.88 (dt, J = 14, 2 Hz, 1H), 8.07 (s,

1H), 8.24−8.21 (d, J = 8.0 Hz, 1H), 8.61−8.56 (m, 1H), 9.83 (bs,

1H). LC-MS (ESI): m/z 298 [M + H]⁺. MP: 135−137 °C.

1-((3,5-Dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbohy-

drazide (4). Ethyl 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyra-

O

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General Procedure B: N-(1-((3,5-Dimethylisoxazol-4-yl)methyl)-

1H-pyrazol-4-yl)-3-hydroxybenzamide (19). The amine hydro-

chloride 6 (227 mg, 1.0 mmol), 3-hydroxybenzoic acid (138 mg,

1.0 mmol), PyBop (624 mg, 1.2 mmol), and triethylamine (0.28 mL,

2.0 mmol) were stirred in dimethylformamide (2 mL) at ambient

temperature for 8 h under a nitrogen atmosphere. The mixture was

diluted with ethyl acetate (3 mL) and washed, successively, with a

saturated NaHCO₃solution (2 × 3 mL) and a saturated NaCl

solution (3 mL). The organic phase was collected, dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated on a rotovap. The

residue was dissolved in methanol (3 mL) and puriﬁed by reverse

phase HPLC (H₂O/acetonitrile, 10/90%). The pure fractions were

concentrated on the rotovap, and the product was recrystallized from

ethanol to aﬀord the title compound 19 as an oﬀ-white solid (180 mg,

(171 mg, 1 mmol), and cesium carbonate (325 mg, 1 mmol) in DMF

(2 mL) was irradiated in the microwave reactor at 85 °C for 20 min.

The reaction mixture was cooled to room temperature, diluted with

aqueous 1 N HCl (100 mL), and extracted with ethyl acetate. The

combined organic extract was dried over sodium sulfate, ﬁltered, and

concentrated. The residue was taken up in methanol (10 mL) and

puriﬁed by reverse phase HPLC to aﬀord the title compound 37 as an

oily material that solidiﬁed upon standing (102 mg, 0.28 mmol, 40%).

¹H NMR (CDCl₃, 400 MHz): δ 2.19 (s, 3H), 2.42 (s, 3H), 3.85 (s,

2H), 4.62 (s, 2 H), 5.06 (s, 2H), 7.40 7.27 (m, 5H), 7.92 (d, J = 0.8

Hz, 1H), 8.08 (d, J = 0.8 Hz, 1H). LC-MS (ESI): m/z 366 [M + H]⁺.

3-(1-(3,5-Dimethylisoxazol-4-yl)methyl-1H-pyrazol-4-yl)-

imidazolidine-2,4-dione (48). The acyl azide 5 (6 g, 25.5 mmol) in

toluene (100 mL) was reﬂuxed for 1 h and cooled to ambient

temperature under a nitrogen atmosphere. Glycine methyl ester-

hydrochloride (3.1 g, 26 mmol) and triethylamine (3.2 g, 32 mmol)

were added, and the mixture was reﬂuxed for 16 h. The solvent was

then removed under a vacuum, and the residue was dissolved in ethyl

acetate (100 mL) and washed twice with a 1 N HCl solution (150

mL). The aqueous phase was back extracted with ethyl acetate (2 ×

75 mL). The combined organic extract was dried over sodium sulfate,

ﬁltered, and concentrated on a rotovap. The resulting solid was

triturated with ethyl acetate/hexanes (1/9) and dried under high

1

0.58 mmol, 58%). H NMR (DMSO-d₆, 400 MHz): δ 2.15 (s, 3H),

2.41 (s, 3H), 5.14 (s, 2H), 6.93−6.95 (m, 1H), 7.27−7.36 (m, 3H),

7.58 (d, J = 0.8 Hz, 1H), 8.09 (d, J = 0.8 Hz, 1H), 9.72 (br s, 1H),

10.34 (br s, 1H). LC-MS (ESI): m/z 313 [M + H]⁺.

General Procedure C: N-(1-((3,5-Dimethylisoxazol-4-yl)methyl)-

1H-pyrazol-4-yl)benzo[d][1,3]dioxole-5-carboxamide (23). A solu-

tion of the amine hydrochloride 6 (7.3 g, 31.7 mmol) in DCM (200

mL) was treated at 0 °C with triethylamine (6.9 g, 68 mmol). The

mixture was stirred until the free amine was completely dissolved.

Benzo[d][1,3]dioxole-5-carbonyl chloride (5.8 g, 32 mmol) in DCM

(50 mL) was then added dropwise. When the addition was complete,

the ice bath was removed, and the reaction mixture was stirred for 1 h.

The mixture was washed with aqueous 1 N HCl (200 mL), and the

aqueous phase was back extracted with DCM (2 × 75 mL). The

combined organic extract was dried over sodium sulfate, ﬁltered, and

concentrated on a rotovap. The crude product was puriﬁed by silica

gel chromatography (60% ethyl acetate in hexanes), and the resulting

light yellow solid was crystallized from ethanol/H₂O to aﬀord the title

compound 23 (4.9 g, 45%) as a white solid. ¹H NMR (DMSO-d₆, 400

MHz): δ 2.15 (s, 3H), 2.41 (s, 3H), 5.14 (s, 2H), 6.12 (s, 2H), 7.04

(d, J = 8.4 Hz, 1H), 7.47 (d, J = 1.6 Hz, 1H), 7.53 (dd, J = 8.4, 1.6 Hz,

1H), 7.57 (d, J = 0.8 Hz, 1H), 8.08 (d, J = 0.8 Hz, 1H), 10.26 (s, 1H).

LC-MS (ESI): m/z 341 [M + H]⁺. MP: 172−173 °C.

1

vacuum to aﬀord 48 as a white solid (5.2 g, 18.9 mmol, 74%). H

NMR (CDCl₃, 400 MHz): δ 2.19 (s, 3H), 2.42 (s, 3H), 4.09 (d, J =

1.2 Hz, 2H), 5.06 (s, 2H), 5.68 (br s, 1H), 7.90 (d, J = 0.8 Hz, 1H),

8.05 (d, J = 0.8 Hz, 1H). LC-MS (ESI): m/z 276 [M + H]⁺.

General Procedure E: 3-(tert-Butyldimethylsilyloxy)-

benzaldehyde (68a). 3-Hydroxylbenzaldehyde 67a (30 g, 0.246

mmol) was disolved in anhydrous DCM (350 mL), and imidazole (3

equiv) was added. The mixture was cooled to 0 °C, and a solution of

tert-butylchlorodimethylsilane (1.3 equiv) in anhydrous DCM (250

mL) was added dropwise. After stirring overnight at room

temperature, the reaction mixture was diluted with DCM (150

mL), washed with a 1 M aqueous HCl solution and brine, and dried

over MgSO₄. After ﬁltration, the solvent was removed under reduced

pressure, and the residue was puriﬁed over silica gel column using 3%

EtOAc in hexanes to give the desired product 68a in a quantitative

yield (57.8 g). ¹H NMR (DMSO-d₆, 400 MHz): δ 0.21 (s, 6H), 0.96

(s, 9H), 7.20 (ddd, J = 1.2, 2.4, 7.6 Hz, 1H), 7.31−7.34 (m, 1H),

7.47−7.56 (m, 2H), 9.96 (s, 1H). LC-MS (ESI): m/z 237 [M + H]⁺.

2,3-Bis((tert-butyldimethylsilyl)oxy)benzaldehyde (68b). 2,3-Di-

hydroxybenzaldehyde 67b (2.76 g, 20 mmol) and tert-butylchlor-

odimethylsilane (15.0 g, 5 equiv) were reacted according to general

2-(1-((3,5-Dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-

isoindoline-1,3-dione (30). 2-(1H-pyrazol-4-yl)isoindoline-1,3-dione

(1.5 g, 7 mmol), 4-(chloromethyl)-3,5-dimethylisoxazole (1.5 g, 10

mmol), and cesium carbonate (3.3 g, 10 mmol) were stirred in DMF

(20 mL) at 80 °C for 3 h. The reaction mixture was cooled to room

temperature, diluted with water (150 mL), and extracted with ethyl

acetate (3 × 75 mL). The combined organic extract was dried over

sodium sulfate, ﬁltered, and concentrated on a rotovap. The solid

product was triturated with ethyl acetate/Hexanes (1/9) and

recrystallized from the reﬂuxing ethanol (30 mL) to aﬀord 30 (900

1

procedure E to aﬀord the title compound 68b (3.77 g, 52%). H

NMR (CDCl₃, 400 MHz): δ 0.15 (s, 6H), 0.24 (s, 6H), 0.98 (s, 9H),

1.03 (s, 9H), 6.92 (br t, J = 8.0 Hz, 1H), 7.08 (dd, J = 2.0, 8.0 Hz,

1H), 7.41 (dd, J = 2.0, 8.0 Hz, 1H). 10.38 (s, 1H). LC-MS (ESI): m/z

367 [M + H]⁺.

1

mg, 38%) as a bright, light yellow solid. H NMR (DMSO-d₆, 400

MHz): δ 2.18 (s, 3H), 2.43 (s, 3H), 5.24 (s, 2H), 7.83 (d, J = 0.8 Hz,

1H), 7.91−7.83 (m, 4H), 8.21 (d, J = 0.8 Hz, 1H). LC-MS (ESI): m/

z 323 [M + H]⁺. MP: 170−171 °C.

General Procedure F: 2-(3-(tert-Butyldimethylsilyloxy)-

benzylamino)acetate (70a). To

a solution of 3-(tert-

N-(1-((3,5-Dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-2-

phenylacetamide (32). A solution of amine hydrochloride 6 (230

mg, 1 mmol) and triethylamine (300 mg, 3 mmol) in DCM (10 mL)

at 0 °C was treated dropewise with 2-phenylacetyl chloride (184 mg,

1.3 mmol). After the addition was complete, the reaction mixture was

stirred for 1 h at room temperature. The mixture was diluted with

DCM (50 mL) and washed with 1 N aqueous HCl (100 mL),

followed by 1 N aqueous NaOH (100 mL) and water (100 mL). The

combined organic extract was dried over sodium sulfate and ﬁltered,

and the solvent was removed under a vacuum. The resulting residue

was puriﬁed by silica gel chromotography (50% ethyl acetate in

hexanes) and triturated in ethyl acetate/hexanes (1/9) to aﬀord

compound 32 as a white solid (188 mg, 68%). ¹H NMR (CDCl₃, 400

MHz): δ 2.15 (s, 3H), 2.38 (s, 3H), 3.69 (s, 2H), 4.96 (s, 2H), 7.15

(br s, 1H), 7.27−7.42 (m, 6H), 7.84 (s, 1H). LC-MS (ESI): m/z 311

[M + H]⁺. MP: 106−108 °C.

butyldimethylsilyloxy)benzaldehyde 68a (19.0 g, 80.38 mol) in

anhydrous DCE (300 mL) were added methyl 2-aminoacetate

hydrochloride 69a (21 g, 16.73 mmol, 2 equiv), 4 Å MS (25 g),

and Et₃N (2.5 equiv). The mixture was stirred for 6 h at room

temperature, cooled to 10 °C, and treated with NaBH(OAc)₃(35 g,

16.51, ∼ 2 equiv) while stirring vigorously. After the addition was

completed, the cooling source was immediately removed, and the

reaction mixture was stirred overnight under nitrogen. The mixture

was quenched with a saturated aqueous NaHCO₃solution, and the

layers were separated. The aqueous phase was further extracted with

DCM (2 × 300 mL). The combined organic extract was washed with

water (50 mL) and brine (100 mL) and dried over MgSO₄. The

solvent was removed under a vacuum, and the residue was puriﬁed

over silica gel using 25% ethyl acetate in hexanes to give the title

1

compound 70a (19.9 g, 64.30 mmol, 78%) as a colorless liquid. H

NMR (DMSO-d₆, 400 MHz): δ 0.17 (s, 6H), 0.95 (s, 9H), 2.47 (br s,

1H), 3.28 (s, 2H), 3.62 (s, 3H), 3.65 (s, 2H), 6.70 (ddd, J = 0.8, 1.2,

8.0 Hz, 1H), 6.81 (m, 1H), 6.89 (dm, J = 7.6 Hz, 1H), 7.17 (pseudo t,

General Procedure D: 1-Benzyl-3-(1-((3,5-dimethylisoxazol-4-

yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione (37). A solution

of compound 48 (200 mg, 0.7 mmol), (bromomethyl)benzene 49

P

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J = 7.6, 8.0 Hz, 1H). LC-MS (ESI): m/z 247.1 [M + H]⁺. HPLC

purity >95%. LC-MS (ESI): m/z 310 [M + H]⁺.

MHz): δ 0.16 (s, 6H), 0.20 (s, 6H), 0.97 (s, 9H), 1.00 (s, 9H), 1.26

(t, J = 7.2 Hz, 3H), 3.36 (s, 2H), 3.73 (s, 2H), 4.16 (q, J = 7.2 Hz,

2H), 6.59 (dd, J = 3.2, 8.4 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 6.74 (d,

J = 3.2 Hz, 1H). LC-MS (ESI): m/z 454 [M + H]⁺.

Ethyl 2-(3-(tert-Butyldimethylsilyloxy)benzylamino)-2-methyl-

propanoate (70b). Similar to general procedure F, to a solution of

3-(tert-butyldimethylsilyloxy)benzaldehyde 68a (48.8 g, 0.206 mol) in

anhydrous DCE (900 mL) were added ethyl 2-amino-2-methyl-

propanoate hydrochloride 69c (86.3 g, 0.515 mol, 2.5 equiv), 4 Å MS

(70 g), and Et₃N (2.5 equiv). The mixture was stirred at room

temperature overnight under nitrogen and then treated with

NaBH(OAc)₃(2 equiv); after the addition was completed, the

reaction mixture was stirred overnight under nitrogen. The mixture

was then diluted with DCM and carefully quenched with a saturated

aqueous NaHCO₃solution, and the layers were separated. The

aqueous phase was further extracted with DCM. The combined

organic extract was washed with water and brine, dried over MgSO₄,

and ﬁltered. The solvent was removed under a vacuum, and the

residue was puriﬁed over silica gel column using EtOAc/hexanes (2/

General Procedure H: 3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-

1H-pyrazol-4-yl)-1-(3-hydroxybenzyl) Imidazolidine-2,4-dione

(S6821). This reaction was performed under N₂. 1-((3,5-Dimethy-

lisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide 5 (38 g, 154

mmol) was stirred at reﬂux in dry toluene (600 mL) along with

molecular sieves until no emission of nitrogen was observed, which

indicates complete conversion of 1-((3,5-dimethylisoxazol-4-yl)-

methyl)-1H-pyrazole-4-carbonyl azide 5 into the 4-((4-isocyanato-

1H-pyrazol-1-yl)methyl)-3,5-dimethylisoxazole intermediate. A solu-

tion of methyl 2-(3-(tert-butyldimethylsilyloxy)benzylamino)acetate

70a (48g, 154 mmol) in dry toluene (100 mL) was prepared. To this

solution, the mixture obtained above, which contains the 4-((4-

isocyanato-1H-pyrazol-1-yl)methyl)-3,5-dimethylisoxazole intermedi-

ate, was added, and the reaction mixture was reﬂuxed for 48 h. The

mixture was then allowed to cool to room temperature and was

ﬁltered over a pad of celite. The ﬁltrate was concentrated on a

rotovap, and the solid residue was suspended in methanol (1 L) and

treated with HCl (2.0 M solution in diethyl ether, 540 mL). The

solution was stirred for 16 h at 50 °C. The reaction mixture was

partially concentrated on a rotovap to remove most of the diethyl

ether, and the mixture diluted with water. The solid that formed was

collected by ﬁltration and recrystallized from hot ethanol (2 L). The

product was collected by ﬁltration and washed with ethanol to aﬀord

S6821 (48 g, 127 mmol, 82%) as a white solid. ¹H NMR (DMSO-d₆,

400 MHz): δ 2.15 (s, 3H), 2.41 (s, 3H), 3.99 (s, 2H), 4.45 (s, 2H),

5.21 (s, 2H), 6.64−6.77 (m, 3H), 7.11−7.19 (m, 1H), 7.80 (d, J = 0.8

Hz, 1H), 8.19 (d, J = 0.4 Hz, 1H), 9.44 (s, H). LC-MS (ESI): m/z

382 [M + H]⁺. EA: calcd. C 59.84, H 5.02, N 18.36; found C 59.79, H

5.41, N 18.42.

1

8) to give the title compound 70b (66.4 g, 92%). H NMR (CDCl₃,

400 MHz): δ 0.19 (s, 6H), 0.98 (s, 9H), 1.30 (t, J = 7.2 Hz, 3H), 1.36

(s, 6H), 3.57 (s, 2H), 4.20 (q, J = 7.2 Hz, 2H), 6.71 (ddd, J = 1.2, 2.4,

8.0 Hz, 1H), 6.82 (m, 1H), 6.93 (dm, J = 7.6 Hz, 1H), 7.16 (pseudo t,

J = 7.6, 8.0 Hz, 1H). LC-MS (ESI): m/z 352 [M + H]⁺.

Ethyl (2,3-Bis((tert-butyldimethylsilyl)oxy)benzyl)glycinate (70c).

2,3-Bis((tert-butyldimethylsilyl)oxy)benzaldehyde 68b (1.50 mg, 4.09

mmol) and ethyl 2-aminoacetate hydrochloride 69b (3 equiv) were

reacted according to general procedure F, and the product was

puriﬁed over silica gel using 20% ethyl acetate in hexanes to aﬀord the

title compound 70c (1.44 g, 3.17 mmol, 76%) as a yellowish oil.

1

Anhydrous THF was used in lieu of DCE as the solvent. H NMR

(DMSO-d₆, 400 MHz): δ 0.10 (s, 6H), 0.20 (s, 6H), 0.90 (s, 9H),

0.97 (s, 9H), 1.17 (t, J = 7.2 Hz, 3H), 2.30 (br s, 1H), 3.25 (s, 2H),

3.67 (s, 2H), 4.06 (q, J = 7.2 Hz, 2H), 6.76 (dd, J = 1.6, 8.0 Hz, 1H),

6.82 (pseudo t, J = 7.6, 8.0 Hz, 1H), 6.97 (dd, J = 1.6, 7.6 Hz, 1H).

LC-MS (ESI): m/z 454 [M + H]⁺.

General Procedure I: 3-(1-((3,5-Dimethylisoxazol-4-yl)methyl)-

1H-pyrazol-4-yl)-1-(3-hydroxybenzyl)-5,5-dimethylimidazolidine-

2,4-dione (S7958). This condition was designed to avoid the isolation

of the acyl azide. Sodium nitrite (17.5 g, 254 mmol) in ice water (390

mL) was added to 1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyra-

zole-4-carbohydrazide 4 (39.8 g, 169 mmol), in 10% acetic acid (1000

mL) and an ice/water bath at a rate that maintained a temperature

below 5 °C. The mixture was stirred for 2.5 h at room temperature

and then extracted twice with DCM (375 mL, 150 mL). The organic

layer was washed with aqueous potassium carbonate (25%, 200 mL),

water (200 mL), and brine and was dried with magnesium sulfate.

The magnesium sulfate was ﬁltered out to give a solution of 1-((3,5-

dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-carbonyl azide 5 in

DCM. The solution was added continuously to a solution of ethyl

2-(3-(tert-butyldimethylsilyloxy)benzylamino)-2-methylpropanoate

70b (59.4 g, 169 mmol) in anhydrous toluene (1300 mL) in the

presence of 4 Å MS (21 g) preheated to 105 °C at a rate that

maintained an internal reaction temperature between 95 and 105 °C.

The DCM was allowed to distill oﬀ during the addition step. The

mixture was then reﬂuxed for 16 h. The MS were ﬁltered out, and the

solvent was removed under a vacuum to give a residue, which was

dissolved in anhydrous methanol (700 mL). HCl was added (2 M in

diethyl ether, 144 mL), and the mixture was heated at 65 °C for 3 h.

The solvent was removed under a vacuum, and the product was

recrystallized from ethanol (330 mL) to give S7958 (63.8 g, 92%) as a

white solide. ¹H NMR (DMSO-d₆, 400 MHz): δ 1.31 (s, 6H), 2.16 (s,

3H), 2.42 (s, 3H), 4.47 (s, 2H), 5.20 (s, 2H), 6.65 (ddd, J = 0.8, 2.4, 8

Hz, 1H), 6.75−6.83 (m, 2H), 7.11 (t, J = 8 Hz, 1H), 7.83 (d, J = 0.4

Hz, 1H), 8.21 (d, J = 0.8 Hz, 1H), 9.36 (s, H). LC-MS: m/z 410 [M

+ H]⁺. EA: calcd. C 61.60, H 5.66, N 17.10; found C 61.69, H 6.05, N

17.32.

General Procedure G: Ethyl (3,4-Bis((tert-butyldimethylsilyl)oxy)-

benzyl)glycinate (70d). 3,4-Dihydroxybenzaldehyde 67c (500 mg,

3.62 mmol) was dissolved in anhydrous DCM (100 mL), and

imidazole (3.5 equiv) was added. The solution was cooled to 0 °C

and treated with small portions of tert-butylchlorodimethylsilane (2.5

equiv). The reaction mixture was warmed slowly to room temper-

ature, stirred overnight, and quenched with a 1 N HCl solution (2

equiv). The organic phase was washed with water and brine, dried

over magnesium sulfate, and ﬁltered. The ﬁltrate was concentrated to

aﬀord a residue containing the protected aldehyde 68c that was used

in the next step without further puriﬁcation. The residue was

dissolved in anhydrous dichloroethane (100 mL), and ethyl 2-

aminoacetate hydrochloride 69b (2.5 equiv) was added, followed by

treatment with TEA (2.5 equiv). Then, 4 Å MS (∼1g) were added,

and the mixture was stirred for 2 h at room temperature. The reaction

mixture was cooled to about 10 °C, and while stirring vigorously,

NaBH(OAc)₃(2 equiv) was added. The reaction mixture was

warmed slowly to room temperature and stirred overnight under

nitrogen. The mixture was quenched with a saturated aqueous

NaHCO₃solution, and the layers were separated. The aqueous phase

was further extracted with DCM (2 × 25 mL). The combined organic

extract was washed with water (25 mL) and brine (50 mL) and dried

over MgSO₄. The solvent was removed under a vacuum, and the

residue was puriﬁed over silica gel using 25% ethyl acetate in hexanes

to aﬀord the title compound 70d (970 mg, 2.14 mmol, 59%, two

steps) as a yellowish oil. ¹H NMR (CDCl₃, 400 MHz): δ 0.18 (s, 6H),

0.19 (s, 6H), 0.98 (s, 18H), 1.27 (t, J = 7.2 Hz, 3H), 3.37 (s, 2H),

3.67 (s, 2H), 4.19 (q, J = 7.2 Hz, 2H), 7.72−7.81 (m, 3H). LC-MS

(ESI): m/z 454 [M + H]⁺.

Ethyl (2,5-Bis((tert-butyldimethylsilyl)oxy)benzyl)glycinate (70e).

2,5-Dihydroxybenzaldehyde 67e (500 mg, 3.62 mmol), tert-

butylchlorodimethylsilane (2.5 equiv), and ethyl 2-aminoacetate

hydrochloride 69b (3 equiv) were reacted according to general

procedure G, and the product was puriﬁed over silica gel using 20%

ethyl acetate in hexanes to aﬀord the title compound 70e (750 mg,

1.65 mmol, 46%, two steps) as a yellowish oil. ¹H NMR (CDCl₃, 400

General Procedure J: 1-(2,5-Dihydroxybenzyl)-3-(1-((3,5-dime-

thylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione

(M397A). 1-((3,5-Dimethylisoxazol-4-yl)methyl)-1H-pyrazole-4-car-

bonyl azide 5 (423 mg, 1.72 mmol) in dry toluene (100 mL) was

treated with 4 Å MS, and the mixture stirred at reﬂux under nitrogen

for 2 h. After cooling the reaction to room temperature, a solution of

Q

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

ethyl (2,5-bis((tert-butyldimethylsilyl)oxy)benzyl)glycinate 70e (910

mg, 2.01 mmol) in dry toluene (20 mL) was added. The mixture was

stirred at room temperature for 2 h, heated to 100 °C, and stirred

overnight. The reaction mixture was cooled to room temperature and

ﬁltered over a pad of celite. The ﬁltrate was concentrated on a

rotovap, and the residue puriﬁed over silica gel using 25% EtOAc in

hexanes to furnish the 1-(2,5-bis((tert-butyldimethylsilyl)oxy)benzyl)-

3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-

imidazolidine-2,4-dione intermediate as a colorless oil (950 mg, 88%)

(LC-MS (ESI): m/z 626 [M + H]⁺). The latter intermediate (850

mg, 1.36 mmol) was then dissolved in methanol (25 mL) and treated

with HCl (2 M in Et₂O, 10 equiv). The reaction mixture was reﬂuxed

for 2 h, and the volatiles were removed under a vacuum. The residual

solid was recrystallized from ethanol to aﬀord the title compound

M397A (295 mg, 0.74 mmol, 54%) as a whitish solid. ¹H NMR

(DMSO-d₆, 400 MHz): δ 2.14 (s, 3H), 2.41 (s, 3H), 4.01 (s, 2H),

4.40 (s, 2H), 5.20 (s, 2H), 6.51 (dd, J = 2.8, 8.4 Hz, 1H), 6.57 (d, J =

3.2 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 0.8 Hz, 1H), 8.19

(d, J = 0.8 Hz, 1H), 8.70 (s, 1H), 8.94 (s, H). LC-MS (ESI): m/z 382

[M + H]⁺. LC-MS (ESI): m/z 398 [M + H]⁺.

gradient to give (2S,3R,4S,5S,6S)-2-(3-((3-(1-((3,5-dimethylisoxazol-

4-yl)methyl)-1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)-

phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triace-

tate intermediate 78 (306 mg, 439 μmol, 18%) as a white solid (LC-

MS (ESI): m/z 698 [M + H]⁺). A solution of the latter

(2S,3R,4S,5S,6S)-2-(3-((3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-

1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)phenoxy)-6-

(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate inter-

mediate (300 mg, 430 μmol) in methanol (5 mL) was treated with

2 N NaOH (10 equiv) and stirred overnight at room temperature.

The reaction mixture was then acidiﬁed to pH 3−4 using a 5 N HCl

solution, stirred for 30 min, and puriﬁed using reverse phase HPLC to

furnish the desired product M557 as a white solid (54 mg, 97 μmol,

23%). ¹H NMR (DMSO-d₆, 400 MHz): δ 2.15 (s, 3H), 2.41 (s, 3H),

3.10−3.54 (m, 3H), 3.89 (d, J = 9.6 Hz, 1H), 4.01 (s, 2H), 4.52 (s,

2H), 5.07 (d, J = 7.6 Hz, 1H), 5.20 (s, 2H), 5.34 (m, 2H), 6.86−7.09

(m, 3H), 7.20−7.40 (m, 1H), 7.80 (d, J = 0.4 Hz, 1H), 8.19 (d, J =

0.8 Hz, 1H), 12.76 (s, 1H). LC-MS (ESI): m/z 558 [M + H]⁺.

ASSOCIATED CONTENT

■

1-(3,4-Dihydroxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)-

methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione (M397B). The

title compound M397B (whitish solid, 300 mg, 0.66 mmol, 46%)

was synthesized according to general procedure J from 70d and the

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00388.

1

azide 5. H NMR (DMSO-d₆, 400 MHz): δ 2.15 (s, 3H), 2.41 (s,

Additional SAR table, experimental details, character-

ization data, and NMR spectral ﬁgures (PDF)

3H), 3.94 (s, 2H), 4.35 (s, 2H), 5.20 (s, 2H), 6.57 (dd, J = 2.0, 8.0

Hz, 1H), 6.67−6.73 (m, 2H), 7.79 (s, 1H), 8.18 (s, H), 8.93 (s, 1H),

8.94 (s, H). LC-MS (ESI): m/z 382 [M + H]⁺. LC-MS (ESI): m/z

398 [M + H]⁺.

Molecular formula strings (CSV)

1-(2,3-Dihydroxybenzyl)-3-(1-((3,5-dimethylisoxazol-4-yl)-

methyl)-1H-pyrazol-4-yl)imidazolidine-2,4-dione (M397C). The

title compound M397C (whitish solid, 675 mg, 1.70 mmol, 62%)

was synthesized according to general procedure J from 70c and the

AUTHOR INFORMATION

■

Corresponding Author

Joseph R. Fotsing − Firmenich SA, R&D North America, San

Diego, California 92121, United States; orcid.org/0000-

0003-4223-7295; Email: joseph.fotsing@ﬁ rmenich.com,

jrfotsing@gmail.com

1

azide 5. H NMR (DMSO-d₆, 400 MHz): δ 2.14 (s, 3H), 2.40 (s,

3H), 3.99 (s, 2H), 4.47 (s, 2H), 5.20 (s, 2H), 6.55−6.67 (m, 2H),

6.75 (dd, J = 2.0, 7.2 Hz, 1H), 7.79 (d, J = 0.4 Hz, 1H), 8.18 (d, J =

0.8 Hz, 1H), 8.55 (br s, 2H). LC-MS (ESI): m/z 382 [M + H]⁺. LC-

MS (ESI): m/z 398 [M + H]⁺.

Authors

Sodium 3-((3-(1-((3,5-Dimethylisoxazol-4-yl)methyl)-1H-pyra-

zol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)phenyl Sulfate (M461

Sodium Salt). A solution of 3-(1-((3,5-dimethylisoxazol-4-yl)-

methyl)-1H-pyrazol-4-yl)-1-(3-hydroxybenzyl) imidazolidine-2,4-

dione S6821 (1.00 g, 2.60 mmol) in anhydrous DCM (50 mL) was

treated with PySO₃(10 equiv), and the mixture was reﬂuxed for 2 h,

cooled to room temperature, and quenched with saturated NaHCO₃

(10 equiv). The mixture was then stirred for 1 h at room temperature,

and MeOH (15 mL) was added. The solid was ﬁltered, and the

organic phase was concentrated. The residue was dissolved in 1/3

MeOH/DCM (30 mL); the solid was ﬁltered, and the ﬁltrate was

concentrated again. The latter was repeated again, and the foamy

residue was dissolved in EtOH and precipitated by the slow addition

of Et₂O. The solid was collected and dried under a vacuum to give the

desired product M461 sodium salt as a white powder (1.20 g, 2.48

Vincent Darmohusodo − Firmenich SA, R&D North America,

San Diego, California 92121, United States

Andrew P. Patron − Firmenich SA, R&D North America, San

Diego, California 92121, United States

Brett W. Ching − Firmenich SA, R&D North America, San

Diego, California 92121, United States

Thomas Brady − Firmenich SA, R&D North America, San

Diego, California 92121, United States

Melissa Arellano − Firmenich SA, R&D North America, San

Diego, California 92121, United States

Qing Chen − Firmenich SA, R&D North America, San Diego,

California 92121, United States

Timothy J. Davis − Firmenich SA, R&D North America, San

Diego, California 92121, United States

Hanghui Liu − Firmenich SA, R&D North America, San Diego,

California 92121, United States

Guy Servant − Firmenich SA, R&D North America, San Diego,

California 92121, United States

Lan Zhang − Firmenich SA, R&D North America, San Diego,

California 92121, United States

Mark Williams − Firmenich SA, R&D North America, San

Diego, California 92121, United States

Michael Saganich − Firmenich SA, R&D North America, San

Diego, California 92121, United States

Tanya Ditschun − Firmenich SA, R&D North America, San

Diego, California 92121, United States

Catherine Tachdjian − Firmenich SA, R&D North America,

San Diego, California 92121, United States

1

mmol, 95%). H NMR (DMSO-d₆, 400 MHz): δ 2.15 (s, 3H), 2.41

(s, 3H), 4.00 (s, 2H), 4.51 (s, 2H), 5.20 (s, 2H), 6.98−7.03 (dm, J =

7.6 Hz, 1H), 7.09−7.16 (m, 2H), 7.26 (br t, J = 8.0 Hz, 1H), 7.79 (d,

J = 0.8 Hz, 1H), 8.20 (d, J = 0.8 Hz, 1H). LC-MS (ESI): m/z 398 [M

+ H]⁺.

(2S,3S,4S,5R,6S)-6-(3-((3-(1-((3,5-Dimethylisoxazol-4-yl)methyl)-

1H-pyrazol-4-yl)-2,4-dioxoimidazolidin-1-yl)methyl)phenoxy)-

3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic Acid (M557). 3-

(1-((3,5-Dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hy-

droxybenzyl) imidazolidine-2,4-dione S6821 (1.00 g, 2.6 mmol) and

(3R,4S,5S,6S)-2-bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-

3,4,5-triyl triacetate 77 (1.34 g, 3.38 mmol, 1.3 equiv) were dissolved

in anhydrous DCM (25 mL), and silver triﬂate (0.97 g, 3.38 mmol,

1.3 equiv) was added. The mixture was stirred overnight in darkness,

and ETA (1 mL) was added. The solution was further stirred for 30

min and ﬁltered through a pad of celite. The ﬁltrate was concentrated,

and the residue was puriﬁed on silica gel using hexanes and a EtOAc

R

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

(9) Zhang, J.; Jin, H.; Zhang, W.; Ding, C.; O’Keeffe, S.; Ye, M.;

Zuker, C. S. Sour Sensing from the Tongue to the Brain. Cell 2019,

179, 392−402.

(10) Zhang, Y.; Hoon, M. A.; Chandrashekar, J.; Mueller, K. L.;

Cook, B.; Wu, D.; Zuker, C. S.; Ryba, N. J. Coding of sweet, bitter,

and umami tastes: different receptor cells sharing similar signaling

pathways. Cell 2003, 112, 293−301.

(11) Margolskee, R. F. Molecular mechanisms of bitter and sweet

taste transduction. J. Biol. Chem. 2002, 277, 1−4.

(12) Saotome, K.; Teng, B.; Tsui, C. C. A.; Lee, W. H.; Tu, Y. H.;

Kaplan, J. P.; Sansom, M. S. P.; Liman, E. R.; Ward, A. B. Structures of

the otopetrin proton channels Otop1 and Otop3. Nat. Struct. Mol.

Biol. 2019, 26, 518−525.

(13) Nelson, G.; Hoon, M. A.; Chandrashekar, J.; Zhang, Y.; Ryba,

N. J.; Zuker, C. S. Mammalian sweet taste receptors. Cell 2001, 106,

381−390.

(14) Chandrashekar, J.; Kuhn, C.; Oka, Y.; Yarmolinsky, D. A.;

Hummler, E.; Ryba, N. J.; Zuker, C. S. The cells and peripheral

representation of sodium taste in mice. Nature 2010, 464, 297−301.

(15) Ishimaru, Y.; Inada, H.; Kubota, M.; Zhuang, H.; Tominaga,

M.; Matsunami, H. Transient receptor potential family members

PKD1L3 and PKD2L1 form a candidate sour taste receptor. Proc.

Natl. Acad. Sci. U. S. A. 2006, 103, 12569−12574.

(16) Horio, N.; Yoshida, R.; Yasumatsu, K.; Yanagawa, Y.; Ishimaru,

Y.; Matsunami, H.; Ninomiya, Y. Sour taste responses in mice lacking

PKD channels. PLoS One 2011, 6, No. e20007.

(17) Tu, Y. H.; Cooper, A. J.; Teng, B.; Chang, R. B.; Artiga, D. J.;

Turner, H. N.; Mulhall, E. M.; Ye, W.; Smith, A. D.; Liman, E. R. An

evolutionarily conserved gene family encodes proton-selective ion

channels. Science 2018, 359, 1047−1050.

(18) Khan, S. I.; Della Santina, C. C.; Migliaccio, A. A. Angular

vestibuloocular reflex responses in Otop1 mice. I. Otolith sensor input

is essential for gravity context-specific adaptation. J. Neurophysiol.

2019, 121, 2291−2299.

Donald S. Karanewsky − Firmenich SA, R&D North America,

San Diego, California 92121, United States

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c00388

Notes

The authors declare the following competing ﬁnancial

interest(s): The authors work or have worked for Firmenich

or its predecessor subsidiaries.

ACKNOWLEDGMENTS

■

The authors thank Mr. Kenneth Simone for his diligence in

getting the lead compounds evaluated by outside collaborators

in formulations containing bitter APIs. The image used in the

abstract graphic was downloaded free of charge from https://

dlpng.com/png/1705614. The authors thank DLPNG for

allowing free download and use of the image.

ABBREVIATIONS USED

■

TASxRy or TxRy, taste receptor y member x; OTOP1,

otopetrin1; PKDxLy, polycystic kidney disease x-like y protein;

ENaC, epithelial sodium channels; CCD, charge-coupled

device; ROI, region of interest; HBSS, Hank’s balanced salt

solution; FLIPR, ﬂuorescent imaging plate reader; dd, doublet

of doublets; dt, doublet of triplets; ddd, doublet of doublets of

doublets; br, broad; DIEA, N,N-diisopropylethylamine; EDCI,

N-ethyl-N′-(3-(dimethylamino)propyl)carbodiimide hydro-

chloride; HOBt, hydroxybenzotriazole; NaBH(OAc)₃, sodium

triacetoxyborohydride; UHT, ultra-high-temperature process-

ing; ANOVA, analysis of variance; 2-AFC, two-alternative

forced choice; CD rat, Sprague−Dawley rat; WHO, World

Health Organization; FAO, Food and Agriculture Organiza-

tion; EFSA, European Food Safety Authority; FEMA, Flavor

and Extract Manufacturers Association; GRAS, generally

recognized as safe

(19) Khan, S. I.; Della Santina, C. C.; Migliaccio, A. A. Angular

vestibuloocular reflex responses in Otop1 mice. II. Otolith sensor

input improves compensation after unilateral labyrinthectomy. J.

Neurophysiol. 2019, 121, 2300−2307.

(20) Adler, E.; Hoon, M. A.; Mueller, K. L.; Chandrashekar, J.; Ryba,

N. J.; Zuker, C. S. A novel family of mammalian taste receptors. Cell

2000, 100, 693−702.

(21) Behrens, M.; Foerster, S.; Staehler, F.; Raguse, J. D.; Meyerhof,

W. Gustatory expression pattern of the human TAS2R bitter receptor

gene family reveals a heterogenous population of bitter responsive

taste receptor cells. J. Neurosci. 2007, 27, 12630−12640.

(22) Chandrashekar, J.; Mueller, K. L.; Hoon, M. A.; Adler, E.; Feng,

L.; Guo, W.; Zuker, C. S.; Ryba, N. J. T2Rs function as bitter taste

receptors. Cell 2000, 100, 703−711.

REFERENCES

■

(1) Chandrashekar, J.; Hoon, M. A.; Ryba, N. J.; Zuker, C. S. The

receptors and cells for mammalian taste. Nature 2006, 444, 288−294.

(2) Chaudhari, N.; Roper, S. D. The cell biology of taste. J. Cell Biol.

2010, 190, 285−296.

(3) Conte, C.; Ebeling, M.; Marcuz, A.; Nef, P.; Andres-Barquin, P.

J. Identification and characterization of human taste receptor genes

belonging to the TAS2R family. Cytogenet. Genome Res. 2002, 98, 45−

53.

(4) Kobayashi, Y.; Habara, M.; Ikezazki, H.; Chen, R.; Naito, Y.;

Toko, K. Advanced taste sensors based on artificial lipids with global

selectivity to basic taste qualities and high correlation to sensory

scores. Sensors 2010, 10, 3411−3443.

(5) Brockhoff, A.; Behrens, M.; Roudnitzky, N.; Appendino, G.;

Avonto, C.; Meyerhof, W. Receptor agonism and antagonism of

dietary bitter compounds. J. Neurosci. 2011, 31, 14775−14782.

(6) Jaggupilli, A.; Singh, N.; De Jesus, V. C.; Gounni, M. S.;

Dhanaraj, P.; Chelikani, P. Chemosensory bitter taste receptors

(T2Rs) are activated by multiple antibiotics. FASEB J. 2019, 33, 501−

517.

(23) Matsunami, H.; Montmayeur, J. P.; Buck, L. B. A family of

candidate taste receptors in human and mouse. Nature 2000, 404,

601−604.

(24) Mueller, K. L.; Hoon, M. A.; Erlenbach, I.; Chandrashekar, J.;

Zuker, C. S.; Ryba, N. J. The receptors and coding logic for bitter

taste. Nature 2005, 434, 225−229.

(25) Brockhoff, A.; Behrens, M.; Massarotti, A.; Appendino, G.;

Meyerhof, W. Broad tuning of the human bitter taste receptor

hTAS2R46 to various sesquiterpene lactones, clerodane and labdane

diterpenoids, strychnine, and denatonium. J. Agric. Food Chem. 2007,

55, 6236−6243.

(26) Fierro, F.; Giorgetti, A.; Carloni, P.; Meyerhof, W.; Alfonso-

Prieto, M. Dual binding mode of “bitter sugars ” to their human bitter

taste receptor target. Sci. Rep. 2019, 9, 8437.

(27) Sharma, P.; Yi, R.; Nayak, A. P.; Wang, N.; Tang, F.; Knight, M.

J.; Pan, S.; Oliver, B.; Deshpande, D. A. Bitter Taste Receptor

Agonists Mitigate Features of Allergic Asthma in Mice. Sci. Rep. 2017,

7, 46166.

(7) Meyerhof, W.; Batram, C.; Kuhn, C.; Brockhoff, A.; Chudoba,

E.; Bufe, B.; Appendino, G.; Behrens, M. The molecular receptive

ranges of human TAS2R bitter taste receptors. Chem. Senses 2010, 35,

157−170.

(8) Teng, B.; Wilson, C. E.; Tu, Y. H.; Joshi, N. R.; Kinnamon, S. C.;

Liman, E. R. Cellular and Neural Responses to Sour Stimuli Require

the Proton Channel Otop1. Curr. Biol. 2019, 29, 3647−3656.

(28) Carey, R. M.; Workman, A. D.; Yan, C. H.; Chen, B.; Adappa,

N. D.; Palmer, J. N.; Kennedy, D. W.; Lee, R. J.; Cohen, N. A.

S

https://dx.doi.org/10.1021/acs.jmedchem.0c00388

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Sinonasal T2R-mediated nitric oxide production in response to

Bacillus cereus. Am. J. Rhinol Allergy 2017, 31, 211−215.

(29) Clark, A. A.; Dotson, C. D.; Elson, A. E.; Voigt, A.; Boehm, U.;

Meyerhof, W.; Steinle, N. I.; Munger, S. D. TAS2R bitter taste

receptors regulate thyroid function. FASEB J. 2015, 29, 164−172.

(30) Dotson, C. D.; Vigues, S.; Steinle, N. I.; Munger, S. D. T1R and

T2R receptors: the modulation of incretin hormones and potential

targets for the treatment of type 2 diabetes mellitus. Curr. Opin

Investig Drugs 2010, 11, 447−454.

(31) Freund, J. R.; Lee, R. J. Taste receptors in the upper airway.

World J. Otorhinolaryngol Head Neck Surg 2018, 4, 67−76.

(32) Hinrichs, A. L.; Wang, J. C.; Bufe, B.; Kwon, J. M.; Budde, J.;

Allen, R.; Bertelsen, S.; Evans, W.; Dick, D.; Rice, J.; Foroud, T.;

Nurnberger, J.; Tischfield, J. A.; Kuperman, S.; Crowe, R.;

Hesselbrock, V.; Schuckit, M.; Almasy, L.; Porjesz, B.; Edenberg, H.

J.; Begleiter, H.; Meyerhof, W.; Bierut, L. J.; Goate, A. M. Functional

variant in a bitter-taste receptor (hTAS2R16) influences risk of

alcohol dependence. Am. J. Hum. Genet. 2006, 78, 103−111.

(33) Maina, I. W.; Workman, A. D.; Cohen, N. A. The role of bitter

and sweet taste receptors in upper airway innate immunity: Recent

advances and future directions. World J. Otorhinolaryngol Head Neck

Surg 2018, 4, 200−208.

(34) Patel, N. N.; Workman, A. D.; Cohen, N. A. Role of Taste

Receptors as Sentinels of Innate Immunity in the Upper Airway. J.

Pathog. 2018, 2018, 9541987.

(35) Lu, P.; Zhang, C. H.; Lifshitz, L. M.; ZhuGe, R. Extraoral bitter

taste receptors in health and disease. J. Gen. Physiol. 2017, 149, 181−

197.

(36) Turner, A.; Veysey, M.; Keely, S.; Scarlett, C.; Lucock, M.;

Beckett, E. L. Interactions between Bitter Taste, Diet and Dysbiosis:

Consequences for Appetite and Obesity. Nutrients 2018, 10, 1336.

(37) Liszt, K. I.; Ley, J. P.; Lieder, B.; Behrens, M.; Stoger, V.;

Reiner, A.; Hochkogler, C. M.; Kock, E.; Marchiori, A.; Hans, J.;

Widder, S.; Krammer, G.; Sanger, G. J.; Somoza, M. M.; Meyerhof,

W.; Somoza, V. Caffeine induces gastric acid secretion via bitter taste

signaling in gastric parietal cells. Proc. Natl. Acad. Sci. U. S. A. 2017,

114, E6260−E6269.

(38) Liszt, K. I.; Hans, J.; Ley, J. P.; Kock, E.; Somoza, V.

Characterization of Bitter Compounds via Modulation of Proton

Secretion in Human Gastric Parietal Cells in Culture. J. Agric. Food

Chem. 2018, 66, 2295−2300.

(39) Lagerstrom, M. C.; Schioth, H. B. Structural diversity of G

protein-coupled receptors and significance for drug discovery. Nat.

Rev. Drug Discovery 2008, 7, 339−357.

(40) Kim, U.; Wooding, S.; Ricci, D.; Jorde, L. B.; Drayna, D.

Worldwide haplotype diversity and coding sequence variation at

human bitter taste receptor loci. Hum. Mutat. 2005, 26, 199−204.

(41) Roudnitzky, N.; Risso, D.; Drayna, D.; Behrens, M.; Meyerhof,

W.; Wooding, S. P. Copy Number Variation in TAS2R Bitter Taste

Receptor Genes: Structure, Origin, and Population Genetics. Chem.

Senses 2016, 41, 649−659.

(42) Roudnitzky, N.; Behrens, M.; Engel, A.; Kohl, S.; Thalmann, S.;

Hubner, S.; Lossow, K.; Wooding, S. P.; Meyerhof, W. Receptor

Polymorphism and Genomic Structure Interact to Shape Bitter Taste

Perception. PLoS Genet. 2015, 11, No. e1005530.

(43) Roudnitzky, N.; Bufe, B.; Thalmann, S.; Kuhn, C.; Gunn, H. C.;

Xing, C.; Crider, B. P.; Behrens, M.; Meyerhof, W.; Wooding, S. P.

Genomic, genetic and functional dissection of bitter taste responses to

artificial sweeteners. Hum. Mol. Genet. 2011, 20, 3437−3449.

(44) Bufe, B.; Breslin, P. A.; Kuhn, C.; Reed, D. R.; Tharp, C. D.;

Slack, J. P.; Kim, U. K.; Drayna, D.; Meyerhof, W. The molecular basis

of individual differences in phenylthiocarbamide and propylthiouracil

bitterness perception. Curr. Biol. 2005, 15, 322−327.

(45) Kim, U. K.; Jorgenson, E.; Coon, H.; Leppert, M.; Risch, N.;

Drayna, D. Positional cloning of the human quantitative trait locus

underlying taste sensitivity to phenylthiocarbamide. Science 2003, 299,

1221−1225.

(46) Pronin, A. N.; Xu, H.; Tang, H.; Zhang, L.; Li, Q.; Li, X.

Specific alleles of bitter receptor genes influence human sensitivity to

the bitterness of aloin and saccharin. Curr. Biol. 2007, 17, 1403−1408.

(47) Soranzo, N.; Bufe, B.; Sabeti, P. C.; Wilson, J. F.; Weale, M. E.;

Marguerie, R.; Meyerhof, W.; Goldstein, D. B. Positive selection on a

high-sensitivity allele of the human bitter-taste receptor TAS2R16.

Curr. Biol. 2005, 15, 1257−1265.

(48) Roura, E.; Aldayyani, A.; Thavaraj, P.; Prakash, S.; Greenway,

D.; Thomas, W. G.; Meyerhof, W.; Roudnitzky, N.; Foster, S. R.

Variability in Human Bitter Taste Sensitivity to Chemically Diverse

Compounds Can Be Accounted for by Differential TAS2R Activation.

Chem. Senses 2015, 40, 427−435.

(49) Bobowski, N.; Reed, D. R.; Mennella, J. A. Variation in the

TAS2R31 bitter taste receptor gene relates to liking for the

nonnutritive sweetener Acesulfame-K among children and adults.

Sci. Rep. 2016, 6, 39135.

(50) Poole, R. L.; Tordoff, M. G. The Taste of Caffeine. J. Caffeine

Res. 2017, 7, 39−52.

(51) Kim, U. K.; Breslin, P. A.; Reed, D.; Drayna, D. Genetics of

human taste perception. J. Dent. Res. 2004, 83, 448−453.

(52) Hayes, J. E.; Wallace, M. R.; Knopik, V. S.; Herbstman, D. M.;

Bartoshuk, L. M.; Duffy, V. B. Allelic variation in TAS2R bitter

receptor genes associates with variation in sensations from and

ingestive behaviors toward common bitter beverages in adults. Chem.

Senses 2011, 36, 311−319.

(53) Drayna, D. Human taste genetics. Annu. Rev. Genomics Hum.

Genet. 2005, 6, 217−235.

(54) Pirastu, N.; Kooyman, M.; Robino, A.; van der Spek, A.;

Navarini, L.; Amin, N.; Karssen, L. C.; Van Duijn, C. M.; Gasparini, P.

Non-additive genome-wide association scan reveals a new gene

associated with habitual coffee consumption. Sci. Rep. 2016, 6, 31590.

(55) Pirastu, N.; Kooyman, M.; Traglia, M.; Robino, A.; Willems, S.

M.; Pistis, G.; d’Adamo, P.; Amin, N.; d’Eustacchio, A.; Navarini, L.;

Sala, C.; Karssen, L. C.; van Duijn, C.; Toniolo, D.; Gasparini, P.

Association analysis of bitter receptor genes in five isolated

populations identifies a significant correlation between TAS2R43

variants and coffee liking. PLoS One 2014, 9, No. e92065.

(56) Wang, X.; Thomas, S. D.; Zhang, J. Relaxation of selective

constraint and loss of function in the evolution of human bitter taste

receptor genes. Hum. Mol. Genet. 2004, 13, 2671−2678.

(57) Glendinning, J. I. Is the bitter rejection response always

adaptive? Physiol. Behav. 1994, 56, 1217−1227.

(58) Lindemann, B. Taste reception. Physiol. Rev. 1996, 76, 719−

766.

(59) Roy, G. M. The applications and future implications of

bitterness reduction and inhibition in food products. Crit. Rev. Food

Sci. Nutr. 1990, 29, 59−71.

(60) Nissim, I.; Dagan-Wiener, A.; Niv, M. Y. The taste of toxicity: A

quantitative analysis of bitter and toxic molecules. IUBMB Life 2017,

69, 938−946.

(61) Drewnowski, A.; Gomez-Carneros, C. Bitter taste, phytonu-

trients, and the consumer: a review. Am. J. Clin. Nutr. 2000, 72,

1424−1435.

(62) Lindemann, B. Receptors and transduction in taste. Nature

2001, 413, 219−225.

(63) Thawabteh, A.; Lelario, F.; Scrano, L.; Bufo, S. A.; Nowak, S.;

Behrens, M.; Di Pizio, A.; Niv, M. Y.; Karaman, R. Bitterless

guaifenesin prodrugs-design, synthesis, characterization, in vitro

kinetics, and bitterness studies. Chem. Biol. Drug Des. 2019, 93, 262.

(64) Baguley, D.; Lim, E.; Bevan, A.; Pallet, A.; Faust, S. N.

Prescribing for children - taste and palatability affect adherence to

antibiotics: a review. Arch. Dis. Child. 2012, 97, 293−297.

(65) Mennella, J. A.; Spector, A. C.; Reed, D. R.; Coldwell, S. E. The

bad taste of medicines: overview of basic research on bitter taste. Clin.

Ther. 2013, 35, 1225−1246.

(66) Brockhoff, A.; Behrens, M.; Niv, M. Y.; Meyerhof, W. Structural

requirements of bitter taste receptor activation. Proc. Natl. Acad. Sci.

U. S. A. 2010, 107, 11110−11115.

T

https://dx.doi.org/10.1021/acs.jmedchem.0c00388

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(67) Greene, T. A.; Alarcon, S.; Thomas, A.; Berdougo, E.; Doranz,

B. J.; Breslin, P. A.; Rucker, J. B. Probenecid inhibits the human bitter

taste receptor TAS2R16 and suppresses bitter perception of salicin.

PLoS One 2011, 6, No. e20123.

(68) Li, J.; Pan, L.; Fletcher, J. N.; Lv, W.; Deng, Y.; Vincent, M. A.;

Slack, J. P.; McCluskey, T. S.; Jia, Z.; Cushman, M.; Kinghorn, A. D.

In vitro evaluation of potential bitterness-masking terpenoids from the

Canada goldenrod (Solidago canadensis). J. Nat. Prod. 2014, 77,

1739−1743.

flavoring substances 26: The 26th publication by the Expert Panel of

the Flavour and Extrct Manufacturers Association provides an update

on recent progress in the consideration of flavouring ingredients

generally recognized as safe under the food additive amendment. Food

Technolology 2013, 67, 38−56.

(84) Hallagan, J. B.; Hall, R. L. Under the conditions of intended use

- New developments in the FEMA GRAS program and the safety

assessment of flavor ingredients. Food Chem. Toxicol. 2009, 47, 267−

278.

(85) JEFCA. Safety Evaluation of certain Food Additives. Seventy-

Sixth Meeting of the Joint FAO/WHO Expert Commitee on Food

Additives. WHO Food Additives Series No.67, 2012; 1−344.

(86) Bolognesi, C.; Castle, L.; Cravedi, J.-P.; Engel, K. H.; Fowler,

P.; Franz, R.; Grob, K.; Guertler, R.; Husoy, T.; Kaerenlampi, S.;

Mennes, W.; Milana, R. M.; Penninks, A.; de Fatima Tavares Pocas,

M.; Silano, V.; Smith, A.; Tlustos, C.; Woelfle, D.; Zorn, H.; Zugravu,

C. A. Scientific opinion on Flavouring Group Evaluation 400

(FGE.400): 3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1 H -pyrazol-4-

yl)-1-(3-hydroxybenzyl)imidazolidine-2,4-dione. EFSA Journal 2016,

14 (7), 4334.

(69) Slack, J. P.; Brockhoff, A.; Batram, C.; Menzel, S.; Sonnabend,

C.; Born, S.; Galindo, M. M.; Kohl, S.; Thalmann, S.; Ostopovici-

Halip, L.; Simons, C. T.; Ungureanu, I.; Duineveld, K.; Bologa, C. G.;

Behrens, M.; Furrer, S.; Oprea, T. I.; Meyerhof, W. Modulation of

bitter taste perception by a small molecule hTAS2R antagonist. Curr.

Biol. 2010, 20, 1104−1109.

(70) Pydi, S. P.; Jaggupilli, A.; Nelson, K. M.; Abrams, S. R.; Bhullar,

R. P.; Loewen, M. C.; Chelikani, P. Abscisic Acid Acts as a Blocker of

the Bitter Taste G Protein-Coupled Receptor T2R4. Biochemistry

2015, 54, 2622−2631.

(71) Pydi, S. P.; Sobotkiewicz, T.; Billakanti, R.; Bhullar, R. P.;

Loewen, M. C.; Chelikani, P. Amino acid derivatives as bitter taste

receptor (T2R) blockers. J. Biol. Chem. 2014, 289, 25054−25066.

(72) Xu, Q.; Singh, N.; Hong, H.; Yan, X.; Yu, W.; Jiang, X.;

Chelikani, P.; Wu, J. Hen protein-derived peptides as the blockers of

human bitter taste receptors T2R4, T2R7 and T2R14. Food Chem.

2019, 283, 621−627.

(73) Zhang, C.; Alashi, A. M.; Singh, N.; Liu, K.; Chelikani, P.;

Aluko, R. E. Beef Protein-Derived Peptides as Bitter Taste Receptor

T2R4 Blockers. J. Agric. Food Chem. 2018, 66, 4902−4912.

(74) Jaggupilli, A.; Howard, R.; Upadhyaya, J. D.; Bhullar, R. P.;

Chelikani, P. Bitter taste receptors: Novel insights into the

biochemistry and pharmacology. Int. J. Biochem. Cell Biol. 2016, 77,

184−196.

(75) Karanewsky, D. S.; Arthur, A. J.; Liu, H.; Chi, B.; Ida, L.;

Markison, S. Toxicological evaluation of two novel bitter modifying

flavour compounds: 3-(1-((3,5-dimethylisoxazol-4-yl)methyl)-1H-

pyrazol-4-yl)-1-(3-hydroxybenzyl)imidazolidine-2,4-dione and 3-(1-

((3,5-dimethylisoxazol-4-yl)methyl)-1H-pyrazol-4-yl)-1-(3-hydroxy-

benzyl)-5,5-dimethylimidazolidine-2,4-dione. Toxicology reports 2016,

3, 310−327.

(76) Frank, O.; Zehentbauer, G.; Hofmann, T. Bioresponse-guided

decomposition of roast coffee beverage and identification of key bitter

compounds. Eur. Food Res. Technol. 2006, 222, 492−508.

(77) Frank, O.; Blumberg, S.; Krumpel, G.; Hofmann, T. Structure

determination of 3-O-caffeoyl-epi-gamma-quinide, an orphan bitter

lactone in roasted coffee. J. Agric. Food Chem. 2008, 56, 9581−9585.

(78) Frank, O.; Zehentbauer, G.; Hofmann, T. Screening and

identification of bitter compounds in roasted coffee brew by taste

dilution analysis. Dev. Food Sci. 2006, 43, 165−168.

(79) Kostenis, E. Is Galpha16 the optimal tool for fishing ligands of

orphan G-protein-coupled receptors? Trends Pharmacol. Sci. 2001, 22,

560−564.

(80) Ashby, J.; Tennant, R. W. Chemical structure, Salmonella

mutagenicity and extent of carcinogenicity as indicators of genotoxic

carcinogenesis among 222 chemicals tested in rodents by the U.S.

NCI/NTP. Mutat. Res., Genet. Toxicol. Test. 1988, 204, 17−115.

(81) Ashby, J.; Tennant, R. W.; Zeiger, E.; Stasiewicz, S.

Classification according to chemical structure, mutagenicity to

Salmonella and level of carcinogenicity of a further 42 chemicals

tested for carcinogenicity by the U.S. National Toxicology Program.

Mutat. Res., Genet. Toxicol. Test. 1989, 223, 73−103.

(82) Tennant, R. W.; Ashby, J. Classification according to chemical

structure, mutagenicity to Salmonella and level of carcinogenicity of a

further 39 chemicals tested for carcinogenicity by the U.S. National

Toxicology Program. Mutat. Res., Rev. Genet. Toxicol. 1991, 257, 209−

227.

(83) Marnett, L. J.; Cohen, S. M.; Fukushima, S.; Goodheram, N. J.;

Hecht, S. S.; Riejtens, I. M. C. M.; Smith, R. L.; Adams, T. B.;

Hallagan, J. B.; Harman, C.; McGowen, M. M.; Taylor, S. V. GRAS

U

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