Pharmacophore-based design of novel oxadiazoles as selective sphingosine-1-phosphate (S1P) receptor agonists with in vivo efficacy

DOI: 10.1002/cmdc.201402557

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Pharmacophore-Based Design of Novel Oxadiazoles as

Selective Sphingosine-1-phosphate (S1P) Receptor

Agonists with in vivo Efficacy

Anna Quattropani,*^[a]Wolfgang H. B. Sauer,^[b]Stefano Crosignani,^[a]Jerome Dorbais,^[a]

Patrick Gerber,^[a]Jerome Gonzalez,^[a]Delphine Marin,^[a]Mathilde Muzerelle,^[a]Fanny Beltran,^[c]

Anthony Nichols,^[c]Katrin Georgi,^[d]Manfred Schneider,^[d]Pierre-Alain Vitte,^[e]Valerie Eligert,^[e]

Laurence Novo-Perez,^[e]Jennifer Hantson,^[e]Sebastien Nock,^[e]Susanna Carboni,^[e]Adriano

Luis Soares de Souza,^[e]Jean-FranÅois Arrighi,^[e]Ursula Boschert,^[e]and Agnes Bombrun^[a]

Sphingosine-1-phosphate (S1P) receptor agonists have shown

promise as therapeutic agents for multiple sclerosis (MS) due

to their regulatory roles within the immune, central nervous

system, and cardiovascular system. Here, the design and opti-

mization of novel [1,2,4]oxadiazole derivatives as selective S1P

receptor agonists are described. The structure–activity relation-

ship exploration was carried out on the three dominant seg-

ments of the series: modification of the polar head group (P),

replacement of the oxadiazole linker (L) with different five-

membered heterocycles, and the use of diverse 2,2’-disubsti-

tuted biphenyl moieties as the hydrophobic tail (H). All three

segments have a significant impact on potency, S1P receptor

subtype selectivity, physicochemical properties, and in vitro ab-

sorption, distribution, metabolism, excretion and toxicity

(ADMET) profile of the compounds. From these optimization

studies, a selective S1P₁agonist, N-methyl-N-(4-{5-[2-methyl-2’-

(trifluoromethyl)biphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzyl)gly-

cine (45), and a dual S1P_1,5agonist, N-methyl-N-(3-{5-[2’-

methyl-2-(trifluoromethyl)biphenyl-4-yl]-1,2,4-oxadiazol-3-yl}-

benzyl)glycine (49), emerged as frontrunners. These com-

pounds distribute predominantly in lymph nodes and brain

over plasma and induce long lasting decreases in lymphocyte

count after oral administration. When evaluated head-to-head

in an experimental autoimmune encephalomyelitis mouse

model, together with the marketed drug fingolimod, a pan-

S1P receptor agonist, S1P_1,5agonist 49 demonstrated compara-

ble efficacy while S1P₁-selective agonist 45 was less potent.

Compound 49 is not a prodrug, and its improved property

profile should translate into a safer treatment of relapsing

forms of MS.

Introduction

Sphingosine-1-phosphate (S1P) receptors form a class of G-pro-

tein-coupled receptors (GPCRs) with five subtypes, denoted as

S1P₁to S1P₅. These receptors are regulated by S1P and have

important regulatory functions in normal physiology and dis-

ease processes, particularly involving the immune, the central

nervous system (CNS), and the cardiovascular system.^[1]Within

the immune system, down regulation of S1P₁prevents the

egress of B and Tcells from lymph nodes (LN) into the lym-

phatic circulation. This is especially relevant in certain autoim-

mune diseases, including multiple sclerosis (MS), in which de-

myelination and brain atrophy occur due to the presence of

autoreactive lymphocytes within the CNS. Accordingly, S1P₁-

directed pharmacologic interventions that aim to retain these

autoreactive lymphocytes in the LN and thus prevent their re-

circulation and subsequent infiltration into the CNS have been

investigated as a means of preventing disease progression in

patients with MS.^[2]S1P receptor agonists have also proven

useful, as shown by sphingolipid-like immunomodulator fingo-

limod (FTY-720, Ia; Figure 1), approved by the US Food and

Drug Administration (FDA) for use in relapsing remitting multi-

ple sclerosis (RRMS) patients since 2010.^[3]

[a] Dr. A. Quattropani,⁺Dr. S. Crosignani, J. Dorbais, Dr. P. Gerber, J. Gonzalez,

D. Marin, Dr. M. Muzerelle, Dr. A. Bombrun

Chemistry Department, Merck Serono S.A.

9 Chemin des Mines, 1202 Geneva (Switzerland)

E-mail: anna.quattropani@asceneuron.com

[b] Dr. W. H. B. Sauer

Computational Chemistry, Merck Serono S.A.

9 Chemin des Mines, 1202 Geneva (Switzerland)

[c] F. Beltran, Dr. A. Nichols

Lead Discovery Technologies, Merck Serono S.A.

9 Chemin des Mines, 1202 Geneva (Switzerland)

[d] Dr. K. Georgi, Dr. M. Schneider

Early ADME, Merck Serono S.A.

9 Chemin des Mines, 1202 Geneva (Switzerland)

[e] Dr. P.-A. Vitte, V. Eligert, L. Novo-Perez, J. Hantson, S. Nock, Dr. S. Carboni,

Dr. A. L. S. de Souza, Dr. J.-F. Arrighi, Dr. U. Boschert

Pharmacology/Neuroimmunology, Merck Serono S.A.

9 Chemin des Mines, 1202 Geneva (Switzerland)

[⁺] Present Address: Asceneuron S.A., EPFL Innovation Park, 1015 Lausanne

Fingolimod is converted in vivo into its phosphate (Ib),

which binds to S1P_1,3ꢀ5. It has been shown to modulate the ac-

tivity of S1P₁in patients with MS and is believed to decrease

immune cell infiltration into the CNS, consistent with its previ-

ously established effects in animal models of the disease.^[4a]

(Switzerland)

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Figure 1. Structures of known S1P₁agonists; I and II require phosphorylation to achieve activity and are administered as prodrugs.

The potential mode of action underlying the therapeutic activi-

ty of fingolimod in RRMS patients is the S1P₁-receptor-depen-

dent inhibition of T and B cell egress from lymph nodes,^[2]al-

though fingolimod, as well as more selective S1P_1,5agonists

such as siponimod (BAF312, III)^[5]and ceralifimod (ONO-4641,

IV),^[6]cross the blood–brain barrier and therefore might also

have additional direct CNS effects.^[4]The S1P₅receptor has

a more restricted expression profile in immune cells and is pre-

dominantly expressed on oligodendrocytes in the white matter

tracts of the CNS, suggesting a potential role in NK cell traffick-

ing and in myelin repair.^[7]Phase 2 clinical trial results of siponi-

mod^[8]and ceralifimod^[9]suggest that S1P₃and S1P₄targeting

are not needed for therapeutic efficacy as measured using

magnetic resonance imaging (MRI). Agonism of S1P₃was

thought to be related to heart and pulmonary side effects in

fingolimod clinical trials,^[3]but S1P_1,5-selective agonist siponi-

mod has shown a similar safety

zoic acid was first selected as a phosphate ester replacement,

due to its druglike properties demonstrated in previous pro-

grams.^[12]Its combination with hydrophobic chains via an oxa-

diazole ring, key linker of SEW2871 (V), a known reference

compound used for in vitro assays, was evaluated.^[13]Privileged

lipophilic chains for S1P mimics included substituted phenyl

with aliphatic, such as isobutyl^[14,15]and cyclohexyl,^[15]and aro-

matic substituents.^[15,16]This article focuses on generic struc-

tures 1 and 2, covering compounds 3–50, with 45 and 49

emerging as front runners, and the ultimate selection of 49 as

a clinical candidate (Figure 2). Structure–activity relationship

(SAR) and optimization studies were focused towards S1P re-

ceptor selectivity, CNS penetration, and lymphopenia in

mouse.

Herein, the rational design and synthesis of a novel oxadia-

zole series as S1P₁agonists are described together with the

profile in clinical trials.^[8b]Never-

theless, extensive research is still

focused on S1P₃-sparing agonists

to avoid potential side effects.^[10]

In the competitive search of

S1P₁receptor agonists, ana-

logues of FTY-720 and FTY-720-

phosphate

monoester

have

been extensively explored in

academia and industry as illus-

trated in Figure 1 by examples

of biological tools compounds,

prodrugs and non-prodrugs.^[11]

In our efforts to find novel

chemical starting points for S1P

receptor drug discovery, we

report herein the design and

evaluation of carboxylic acid de-

rivatives of oxadiazoles as selec-

tive non-prodrug S1P receptor

agonists.

Our investigation was built on

our internal knowledge of phos-

phate ester isosteres. Fluoroben- Figure 2. S1P₁agonists disclosed in this article with 1 and 2 as generic structure.

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identified SAR, the physicochemical properties, the in vitro

absorption, distribution, metabolism, and excretion (ADME)

properties, and the results of pharmacological evaluation in

a lymphopenia pharmacokinetic /pharmacodynamic (PK/PD)

model. Selected compounds were further assessed, based on

selectivity and safety, in vivo in a myelin oligodendrocyte gly-

coprotein peptide (MOG_35–55)-induced experimental autoim-

mune encephalomyelitis (EAE) murine disease model relevant

for the treatment of MS.

pared from the addition of aqueous hydroxylamine to aromatic

nitrile 51. The hydrophobic tail (H) consisted of a phenyl sub-

stituted with aliphatic (e.g., cyclohexyl, isobutyl) or aromatic

groups; starting materials that were not commercially available

were synthesized independently. In particular, the 2,2’-disubsti-

tuted bis-aryl carboxylic acid derivatives (56) were prepared by

palladium-catalyzed Suzuki cross-coupling reactions, followed

by ester saponification. Subsequent 1,2,4-oxadiazole formation

provided a series of derivatives 59, leading to compounds 3–

14 after saponification. Diverse conditions for oxadiazole for-

mation were used successfully in the progression of the proj-

ect.^[19]The optimal conditions that were applicable from milli-

gram to multigram scale involved 1-[bis(dimethylamino)methy-

lene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluoro-

phosphate (HATU)-mediated coupling of carboxylic acid 56

with amidoxime 52a at room temperature in N,N-dimethyl-

formamide (DMF). The cyclization of intermediate 58 was then

performed either by conventional heating overnight or under

microwave irradiation for 30 min.

Rational Design and Synthesis

Starting from compounds 1, mimics of the lipohilic chain of

sphingosine were explored, maintaining the fluorobenzoic acid

head group and oxadiazole linker. Our aim was to optimize the

potency on the S1P₁receptor, the selectivity versus the S1P₃

receptor, as well as overall druglike properties for oral absorp-

tion and CNS exposure. Computed property ranges in line with

good oral bioavailability^[17]and CNS exposure^[18]were used to

guide our optimization, in particular overall lipophilicity calcu-

lated at pH 7.4 (cLogD_7.4<3), topological polar surface area

(TPSA) (<90 ꢁ²), number of hydrogen-bond donors (HBD=1–

2) and basicity for the amino-containing compounds (pK_a<8).

A simple synthetic strategy in hand (Scheme 1) allowed us

to explore the SAR of compounds 1. The general synthetic

method used for the preparation of the oxadiazole fluoroben-

zoic acid derivatives (1) was the coupling and cyclization of an

amidoxime and a carboxylic acid. Amidoxime 52a was pre-

In the synthesis of 2,2’-disubstituted bis-aryl carboxylic acid

56, all aryl bromide and boronic acids were commercially avail-

able, except for acids 56a,b,c, where the aryl bromides needed

to be prepared (Scheme 2). For the preparation of compounds

56a and 56b, methyl 4-bromo-3-(methoxymethyl)benzoate

53c was obtained in two steps, benzylic bromination of

methyl 4-bromo-3-methylbenzoate 53a followed by its substi-

tution with methanol. Compounds 56a and 56b were ob-

tained after two additional steps. Compound 56c was pre-

Scheme 1. General synthetic scheme for the preparation of 1,2,4-oxadiazole flurorobenzoic acid analogues 1. Reagents and conditions: a) H₂NOH in 50% H₂O,

EtOH, RT, 15 h, 90%; b) Pd(PPh₃)₄, K₂CO₃, toluene/H₂O (1:1), 1108C, 1–15 h, 59–95%; c) NaOH or LiOH, THF/H₂O (1:1), between RT and 1008C, 1–15 h, 59–95%;

d) Optimal conditions: HATU, DIEA, DMF, 08C!RT, 2–3 h, quantitative; e) toluene/DMF (1:1), 958C, 15 h or CH₃CN, 1508C, microwave irradiation (full power,

400 W), 30 min, 34–85%; f) aq NaOH, THF/MeOH (1:1), RT, 1–15 h, 68–98%.

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Scheme 2. Synthesis of 2,2’-disubstituted bis-aryl carboxylic acid 56a–c. Reagents and conditions: a) NBS, AIBN, CH₃CN, reflux, 2 d, 62%; b) MeOH, reflux, 4 d,

94%; c) when R=Me: o-tolylboronic acid; when R=CF₃: 2-(trifluoromethyl)phenylboronic acid, Pd(PPh₃)₄, K₂CO₃, toluene/H₂O (1:1), 1108C, 1–15 h, 63–95%;

d) NaOH, EtOH/H₂O (1:1), 608C, 1 h, 74–92%; e) EtBr, K₂CO₃, CH₃CN, 508C, 2 d, 98%; f) LiOH, THF/H₂O (1:1), RT, 15 h, 93%.

pared from methyl 4-bromo-3-hydroxybenzoate 53d. After

Suzuki cross-coupling with ortho-tolylboronic acid, phenol alky-

lation with ethyl bromide was performed in acetonitrile in the

presence of potassium carbonate. Saponification of the methyl

ester gave 56c that was used in the synthesis of the oxadia-

zole.

2’-substituent of the second phenyl, kept in a favorable confor-

mation by the 2-substituent of the first; 2) The distance be-

tween the hydrophobic and the acceptor feature of the head

group (P); for the fluorobenzoic acid, originally selected as

phosphate monoester mimetic, this distance was identified as

being too great to be covered by the aromatic ring and a car-

boxyl oxygen. This led to the exploration of diverse spacers be-

tween the two parts of P (compounds 15–28).^[21]To this end,

three spacers (namely ether, amine and amide) between the

carboxyl groups and the aromatic nitrile were explored

(Scheme 3). Some oxygen-containing spacers were evaluated

first. Phenoxyacetic acid tert-butyl ester analogue 61 was pre-

pared by O-alkylation of 3-fluoro-4-hydroxy-benzonitrile 60

with tert-butyl bromoacetate in the presence of cesium car-

bonate. Benzyl ether analogues 63 were prepared by alkylation

of corresponding benzyl alcohol 62 (X=OH) or by addition of

2-tert-butyl glycolate to benzyl bromide 62 (X=Br) under

phase-transfer conditions (Scheme 3).^[22]

To support our medicinal chemistry progression, a pharmaco-

phore model was constructed using Catalyst HipHop^[20]from

nine diverse S1P₁ligands from the literature and internal

screening. Five common pharmacophoric features were identi-

fied (Figure 3): three hydrophobic motifs and two hydrogen-

bond acceptors (shown with the putative donor locations on

the receptor). Their geometric arrangement is described in the

Experimental Section.

This model provided two important hints for modifications

around compounds 1: 1) The orientation of the hydrophobic

centers of the hydrophobic tail (H), which could be filled opti-

mally with a 2,2’-disubstitued biphenyl, one by the interior aro-

matic ring and the other (terminal) hydrophobic center by the

Nitrogen-containing spacers were evaluated next. To access

polysubstituted head groups (R³¼H), several benzyl bromide

reagents were prepared by radical bromination of the corre-

sponding methyl-substituted aromatic nitrile precursor using

N-bromosuccinimide (NBS) in the presence of a,a’-azoisobutyr-

onitrile (AIBN). The resulting benzyl bromide derivatives (65)

were reacted with sarcosine tert-butyl ester hydrochloride or

tert-butyl N-methyl-b-alaninate^[23]in the presence of potassium

carbonate. The same synthetic pathway could be applied suc-

cessfully for the synthesis of pyridine equivalent 52p. For the

synthesis of secondary amines analogues, tert-butyl glycinate

was added to benzyl bromide 67. The resulting secondary

amine was protected with a tert-butyloxycarbonyl (boc) group

that was maintained until the end of the synthesis. These dif-

ferent aromatic nitriles were transformed into the correspond-

ing amidoximes (52b–u), which were used in the synthesis of

the 1,2,4-oxadiazole derivatives. For some analogues (70), cycli-

zation at 1508C was in competition with the cleavage of the

oxime bond, yielding the liberation of carboxylic acid 56 and

Figure 3. Compound 1 mapped onto a pharmacophore model derived from

literature and in-house data on S1P₁modulator characteristics; correspond-

ence to the hydrophobic tail (H), oxadiazole linker (L) and polar head group

(P) as indicated.

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Scheme 3. Reactions of substituted aromatic nitrile as head group precursors. Reagents and conditions: a) tert-butyl bromoacetate, Cs₂CO₃, CH₃CN, RT, 15 h,

97%; b) H₂NOH in 50% H₂O, EtOH, RT, 15 h, 77–97%; c) when X=OH: tert-butyl bromoacetate; when X=Br: 2-tert-butyl glycolate, Bu₄N SO₄H, aq NaOH, tolu-

ene, RT, 15 h, 60–70%; d) NBS, AIBN, CH₃CN, reflux, 15 h, 65–75%; e) sarcosine tert-butyl ester hydrochloride or N-methyl-b-alaninate, K₂CO₃, CH₃CN, RT, 15 h,

85–98%; f) tert-butyl glycinate, K₂CO₃, CH₃CN, RT, 15 h, 60–86%; g) Boc₂O, DIEA, CH₂Cl₂, 3.5 h, 75–77%; h) Optimal conditions: HATU, DIEA, DMF, 08C!RT, 2–

3 h, quantitative; i) toluene/DMF (1:1), 958C, 15 h or CH₃CN, 1508C, microwave irradiation ( full power, 400 W), 30 min, 34–85%; j) when R=Me or Et: aq

NaOH, THF/MeOH (1:1), RT, 1–15 h, 68–98%; when R=tBu: HCl, dioxane, RT, 15 h, 54–95%.

the amidine equivalent of 52, as reported under hydrogena-

tion conditions.^[24]This could be prevented by performing this

step at lower temperature (958C) and longer reaction time of

15 hours.

tency, selectivity and importantly physical and pharmaceutical

profile.^[25]Regioisomers of oxadiazoles, triazoles and thiadia-

zoles were synthesized. The alternative 1,2,4-oxadiazole iso-

mers such as compound 29 were prepared from benzoic acid

73 and amidoxime 75. Addition of sarcosine tert-butyl ester to

3-(bromomethyl)benzoic acid 72 gave compound 73. Inde-

pendently, 56a was transformed into the corresponding aro-

matic nitrile 74 in three steps: acyl chloride formation, ammo-

nia addition, and dehydration of the resulting primary amide.

Addition of aqueous hydoxyamine yielded amidoxime 75. Pre-

viously described conditions for the formation of the 1,2,4-oxa-

diazole and tert-butyl ester deprotection gave compound 29.

Acyl hydrazone 77 was obtained by coupling of 56a with boc-

Furthermore, the amide bond was assessed as a spacer.

Amide derivatives 15–18 were prepared by coupling the acid

chloride of compound 13 with diverse amino esters

(Scheme 4). The last step consisted of ester hydrolysis per-

formed either under basic conditions for methyl and ethyl

esters or under acidic conditions for tert-butyl esters.

Although the 1,2,4-oxadiazole heterocycle was first used for

direct comparison with known S1P₁agonists, the replacement

of the oxadiazole linker (L) was obviously challenged for po-

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in potency was obtained with a 2,2’-disubstitution as in com-

pounds 8–14, leading to K_ivalues below 50 nm.

Mapping of early compounds onto the pharmacophore

matched the distal hydrophobic feature against the ortho sub-

stituent of the terminal phenyl ring. This required the angle

between the two rings to be at least 708, which led us to con-

centrate on 2,2’-substituted systems.^[27]The SAR showed that

electron-donating (e.g., OMe) and electron-withdrawing (e.g.,

CF₃) groups were tolerated in both positions (see compounds

9–12). Further elaboration around compound 9, with the intro-

duction of a distal ether group, yielded compound 13 with po-

tency below 10 nm and greater than 700-fold selectivity for

S1P₁over S1P₃. For this compound 13, cLogD_7.4and TPSA

values were 1.9 and 85.5 ꢁ², respectively, which is in the desira-

ble range for a CNS drug.^[18]The position of the ether function-

ality was probed by moving it to the 2’-position of the biphen-

Scheme 4. Synthesis of amides 15–18. Reagents and conditions: a) oxalyl

chloride, DMF (cat), CH₂Cl₂, 08C!RT, 2 h, quantitative; b) HNR¹(CH₂)_nCOOMe,

DIEA, THF, RT, 15 h, 36–63%; c) aq NaOH, THF/MeOH (1:1), RT, 1–3 h, 52–

97%.

Table 1. Structure–activity relationships of fluoro benzoic acid analogues 3–14.^[a]

protected hydrazine, followed by boc deprotection.

It was then coupled with acid 73 by using HATU as

a coupling agent, yielding 78, which was used as

a common intermediate for the synthesis of 1,3,4-

oxadiazole 30 and 1,3,4-thiadiazole 31. Compound

30 was obtained by treatment of 78 with anhydride

trifluoroacetate and 1-methylimidazole, followed by

ester hydrolysis. Reaction of 78 with Lawesson’s re-

agent at 1208C under microwave irradiation gave

compound 31 after ester hydrolysis. Triazole 81 was

prepared from acyl hydrazone 77 and aromatic ni-

trile 80 at 2008C under microwave irradiation in the

presence of potassium carbonate. Compound 32

was isolated after hydrolysis of the tert-butyl ester.

With the different synthetic pathways described

above, all combinations incorporating hydrophobic

biphenyls as H, heterocycles as L, and another hydro-

phobic site substituted by a polar head group for P,

are accessible, enabling us to optimize this novel

chemical series regarding S1P₁potency, S1P receptor

selectivity, ADME and safety profile.

Compd

R¹

R

K_i[nm]

cLogD_7.4TPSA

S1P₁

S1P₃

[ꢁ²]

3

4

5

6

7

8

9

10

11

12

13

14

H

Me

H

iso-Bu

Cy

Ph

460ꢁ57

145ꢁ21

>20000

4050ꢁ1600

>20000

1.5

2.1

1.5

2.2

2.0

2.8

1.7

2.9

1.9

2.9

1.9

76.2

85.5

76.2

85.5

76.2

85.5

4900ꢁ4800

90.5ꢁ4.9

530ꢁ99

Ph

2-Me-Ph

2-OMe-Ph

2-CF₃-Ph

Me

28.5ꢁ3.5 4950ꢁ1800

OMe

CF₃

Me

28.5ꢁ0.7

43ꢁ1.4

>20000

2300ꢁ420

>20000

31.5ꢁ3.5

22ꢁ5.7

Me

>20000

CH₂OMe 2-Me-Ph

Me 2-CH₂OMe-Ph

2.9ꢁ0.2 2200ꢁ280

145ꢁ7.1

>20000

[a] K_ivalues are the mean ꢁSD of three replicates and a minimum of two independ-

ent experiments. Calculated overall lipophilicity at pH 7.4 (cLogD_7.4) and topological

polar surface area (TPSA).

Structure–Activity Relationship Study

yl moiety, yielding 14; this modification resulted in a substantial

All compounds synthesized in this program were first tested in

radioligand binding assays on S1P₁and S1P₃, to optimize the

potency on S1P₁and the selectivity versus S1P₃. In the first

steps of optimization, the lipophilic part of the phenyl-

[1,2,4]oxadiazole fluorobenzoic acid derivatives was optimized

(Table 1). As reported, the n-octyl chain at the phenyl ring of

fingolimod can be replaced by a wealth of lipophilic resi-

dues.^[26]In our series, alkyl substituents show only moderate

activity (data not shown), and the isobutyl substituent (com-

pound 3) was one of the best. Better potency was obtained

with a cyclohexyl group (compound 4). Its replacement by

a phenyl group significantly decreased the potency (cf., com-

pound 5). The introduction of substituents in the 2 or 2’ posi-

tion of the biphenyl moiety allowed recovery of the initial po-

tency of 3 and 4 (cf., compounds 6 and 7). The breakthrough

drop in S1P₁potency. Other modifications in the substitution

of the biphenyl moiety of the [1,2,4]oxadiazole fluorobenzoic

acid series did not yield an improvement in potency on S1P₁

or selectivity over S1P₃, confirming compound 13 as the most

potent, with more than 700-fold selectivity versus S1P₃.

The choice of diverse spacers (ether, amines and amides)

was guided and filtered by the pharmacophore hypothesis

presented in Figure 3; the results are summarized in Table 2. In

the exploration of an alternative to the fluorobenzoic acid ini-

tially used as a head group, fluorobenzoic amide analogues

were explored first as straightforward modifications. The dis-

tance defined by the number of methylene groups between

the carboxylic acid group and the fluorobenzoic amide func-

tionality was varied incrementally to optimize S1P₁binding po-

tency and S1P₃selectivity (cf., 15–17). The length of the chain

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respectively, with comparable

or slightly higher cLogD_7.4

values versus fluorobenzoic

acid analogues, TPSA values of

89 ꢁ², and calculated pK_afor

the tertiary amines of 7.7 and

8.3, respectively. As expected

from the pharmacophore hy-

Table 2. Structure–activity relationships of the spacer between the hydrophobic site and the polar head group

(P) in derivatives 15–28.^[a]

Compd

R¹

R²

K_i[nm]

cLogD_7.4

basic

TPSA

[ꢁ²]

pothesis,

ortho-substituted

S1P₁

S1P₃

pK_a

amino acid 28 was much less

active on S1P₁.

15

16

17

18

19

20

21

22

23

24

25

26

27

28

4-CONHCH₂COOH

3-F

H

2.08ꢁ0.22

1.71ꢁ0.09

2.7ꢁ0.9

440ꢁ270

186ꢁ30

445ꢁ64

177ꢁ120

270^[b]

0.8

1.5

1.6

1.4

1.0

0.7

1.2

3.1

2.0

2.7

3.8

2.8

3.4

2.7

–

8.0

7.7

7.8

8.3

8.0

8.4

114.6

105.8

94.7

97.5

88.7

97.5

88.7

4-CONH(CH₂)₂COOH

4-CONH(CH₂)₃COOH

4-CON(CH₃)(CH₂)₂COOH

4-OCH₂COOH

4-CH₂OCH₂COOH

3-CH₂OCH₂COOH

4-CH₂NHCH₂COOH

4-CH₂N(CH₃)CH₂COOH

4-CH₂N(CH₃)(CH₂)₂COOH

3-CH₂NHCH₂COOH

3-CH₂N(CH₃)CH₂COOH

3-CH₂N(CH₃)(CH₂)₂COOH

2-CH₂N(CH₃)CH₂COOH

The replacement of the cen-

tral [1,2,4]oxadiazole by alter-

native five-membered ring het-

erocycles was evaluated with

the synthesis of a small set of

compounds 29–32 (Scheme 5).

These analogues showed an

order of magnitude lower po-

tency compared with com-

pound 26 (Table 3). Surprising-

ly, none of these analogues ex-

hibited a decrease in lipophilic-

ity. In particular, the shift in

lipophilicity of the [1,3,4]-oxa-

diazole compound 30 versus

10.5ꢁ5.0

1.93ꢁ0.31

1.2^[b]

440^[b]

0.81ꢁ0.19

0.35ꢁ0.23

0.40ꢁ0.24

0.23ꢁ0.14

0.76ꢁ0.05

0.27ꢁ0.12

0.24ꢁ0.01

107ꢁ74

1040ꢁ85

200ꢁ73

865ꢁ520

108ꢁ7

25.5ꢁ0.7

75.3ꢁ6.2

25.5ꢁ0.7

>20000

H

[a] K_ivalues are the mean ꢁSD of three replicates and a minimum of two independent experiments. Calculated

overall lipophilicity at pH 7.4 (cLogD_7.4), bacicity of amino-containing compounds (pK_a) and topological polar

surface area (TPSA). [b] Value corresponds to the mean of three replicates of a single experiment.

(1–3 carbon atoms) had no real impact on S1P₁potency, with

all compounds exhibiting S1P₁activity close to that of com-

pound 13, and yielded a drop in selectivity over S1P₃of

a factor ranging from 100- to 300-fold. To decrease

the number of hydrogen-bond-donor groups, N-al-

kylation of 16 was undertaken, yielding 18, which

exhibited decreased S1P₁potency compared with

16. All fluorobenzoic amide analogues possess a cal-

culated TPSA greater than 90 ꢁ², encouraging us to

explore alternative linkers.

compound 26 was opposite to the reported impact of such an

isomer on the lipophilicity.^[25]These results encouraged us to

keep the [1,2,4]oxadiazole as the central core.

Table 3. Structure–activity relationships of the linker heterocycle (Het) in derivatives

29–32.^[a]

Ether derivatives such as 2-fluoro-phenoxyacetic

acid combined with the lipophilic part of 13 yielded

19, with similar S1P₁potency. The shift of the heter-

oatom to the benzylic position improved S1P₃selec-

tivity (cf. 20 and 21 vs 19); more than 1000-fold se-

lectivity could be obtained with the head group in

the meta position (compound 21). Replacement of

the ether by an amine yielded an improvement in

S1P₁potency with K_ivalues below 0.5 nm (cf., 22–

27). Secondary or tertiary benzylic amines substitut-

ed with acetic or propionic acids exhibited similar

binding potency on S1P₁. On the other hand, such

modifications also had a significant impact on S1P₃

selectivity. The best S1P₃selectivity was obtained

with tertiary benzylic amines, substituted with an

acetic acid, as illustrated with 23 versus 22 and 24,

and 26 versus 25 and 27. In contrast to the benzylic

ethers, amino acids gave better selectivity in the

para rather than the meta position (cf. 23 vs 26).

Compounds 23 and 26 were characterized as having

S1P₃selectivity of greater than 2000- and 280-fold,

Compd

29

Het

K_i[nm]

cLogD_7.4

basic

pK_a

TPSA

[ꢁ²]

S1P₁

S1P₃

1.49ꢁ0.11

2.19ꢁ0.61

0.78ꢁ0.40

3.87ꢁ1.30

470ꢁ42

2.8

8.4

8.6

88.7

75.5

91.3

30

>20000

395ꢁ7

3.5

31

4.0

32

5900ꢁ283

3.3

[a] K_ivalues are the mean ꢁSD of three replicates and a minimum of two independ-

ent experiments. Calculated overall lipophilicity at pH 7.4 (cLogD_7.4), bacicity of

amino-containing compounds (pK_a) and topological polar surface area (TPSA).

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Scheme 5. Synthesis of alternative 5-membered ring heterocycle L. Reagents and conditions: a) sarcosine tert-butyl ester hydrochloride, K₂CO₃, CH₃CN, 608C,

6 h, 97%; b) oxalyl chloride, DMF (cat), CH₂Cl₂, 08C!RT, 3 h, quantitative; c) 0.5m NH₃in dioxane, toluene, RT, 15 min, 91%; d) (CF₃CO)₂O, pyridine, dioxane,

RT, 3 h, 67%; e) 50% NH₂OH in H₂O, EtOH, RT, 15 h, 90%; f) 1. HATU, DIEA, DMF, RT, 2 h; 2. toluene/DMF (1:1), 958C, o/n, or CH₃CN, 1508C, microwave irradia-

tion (full power, 400 W), 30 min, 36%; g) HCl, dioxane, RT, 2–15 h, 68–88%; h) oxalyl chloride, DMF (cat), toluene, 08C!RT, 3 h, quantitative; i) H₂NNHBoc,

DIEA, CH₂Cl₂, RT, 15 h, quantitative; j) TFA, CH₂Cl₂, RT, 5 h, 98%; k) HATU, DIEA, DMF, RT, 3 h, 35%; l) when Q=O: (CF₃CO)₂O, 1-methylimidazole, CH₂Cl₂, RT, 2 h,

69%; when Q=S: Lawesson’s reagent, THF, 1208C, microwave irradiation (full power, 400 W), 30 min, 52%; m) K₂CO₃, nBuOH, 2008C, microwave irradiation

(full power, 400 W), 2 h, 15%.

A last set of compounds (Table 4) explored modifications of

the lipophilic part and substitution of the head group, keeping

the tertiary benzylic amine substituted with an acetic acid as

phosphate mimetic as in compounds 23 and 26. Starting from

compound 23, small lipophilic substituents were tolerated on

the head group, such as a fluoro or a trifluoromethyl group

(see compounds 33–36). These groups yielded a slight increase

in S1P₁potency without considerable impact on the basicity of

the tertiary amine, with calculated pK_avalues ranging from 7.7

to 8.1. Pyridine analogue 37, selected as a bioisosteric replace-

ment of 3-fluoro analogue 33, showed a significant decrease in

lipophilicity and tertiary amine basicity, but a high TPSA, cou-

pled with a drop in S1P₁potency.

the best tolerated regarding S1P₁potency, with compound 40

characterized by a K_ivalue of 0.2 nm but only 112-fold selectivi-

ty versus S1P₃. Its replacement with a trifluoromethyl group

yielded compound 42, which exhibited a one-log drop in S1P₁

potency and an important increase in lipophilicity with

a cLogD_7.4value of 3.8 versus 2.8 for compound 26.

Further elaboration of the hydrophobic tail was explored

with compounds 43–50. Displacement of the benzylic ether to

the phenolic position led to compound 43 with potency com-

parable with compound 23 but poorer S1P₃selectivity. The in-

troduction of the trifluoromethyl group on the biphenyl

moiety, as exemplified in compound 44 with a cLogD_7.4value

of 2.2, had less impact on the lipophilicity than on the head

group (cf. compound 42). Its S1P₁potency and S1P₃selectivity

were comparable with those of compound 23. Further im-

provement in S1P₃selectivity was obtained by removing the

ether moiety, resulting in compounds 45 and 46 with S1P₃K_i

For the meta-substituted series, starting with compound 26,

the impact of a fluorine substituent on S1P₁potency and S1P₃

selectivity was probed by moving it around the aromatic head

group (see compounds 38– 41). Substitution on position 5 was

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Table 4. Structure–activity relationships around compounds 23 and 26, with the modifications of the lipophilic part and substitution of the head group,

and the impact of these structural modification on the physicochemical properties of the resultant derivatives 33–50.^[a]

Compd

R¹

R²

X

R³

R⁴

K_i[nm]

cLogD_7.4

basic

pK_a

TPSA

[ꢁ²]

S1P₁

S1P₃

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

4-CH₂N(CH₃)CH₂COOH

3-CH₂N(CH₃)CH₂COOH

4-CH₂N(CH₃)CH₂COOH

3-CH₂N(CH₃)CH₂COOH

3-CH₂OCH₂COOH

3-F

CH

N

CH₂OMe

OCH₂Me

CH₂OMe

Me

CF₃

Me

0.22ꢁ0.12

0.33ꢁ0.06

0.89ꢁ0.16

0.49ꢁ0.01

1.93ꢁ0.65

0.27ꢁ0.04

0.75ꢁ0.28

0.20ꢁ0.07

1.86ꢁ0.12

2.96ꢁ0.26

0.31ꢁ0.15

0.66ꢁ0.06

1.09ꢁ0.38

0.84ꢁ0.08

0.42ꢁ0.12

0.71ꢁ0.21

0.48ꢁ0.21

0.98ꢁ0.14

128ꢁ27

320ꢁ42

2.1

1.5

2.9

0.6

2.4

3.0

2.6

2.4

3.8

2.4

2.2

3.0

2.8

3.0

3.1

3.8

2.2

7.8

7.9

8.0

8.1

7.2

8.2

8.1

8.4

8.2

7.7

7.6

7.7

7.8

8.3

–

88.7

101.6

88.7

79.5

85.5

2-F

3-CF₃

2-CF₃

H

2-F

4-F

5-F

6-F

5-CF₃

H

3-F

H

1500ꢁ490

1800ꢁ420

1650ꢁ210

140ꢁ13

CH

220^[b]

22.2ꢁ0.4

1550ꢁ71

7550ꢁ2190

244ꢁ17

1400ꢁ170

>20000

Me

CF₃

>20000

847ꢁ230

470ꢁ160

252ꢁ93

603ꢁ24

CF₃

[a] K_ivalues are the mean ꢁSD of three replicates and a minimum of two independent experiments. Calculated overall lipophilicity at pH 7.4 (cLogD_7.4), ba-

cicity of amino-containing compounds (pK_a) and topological polar surface area (TPSA). [b] Value corresponds to the mean of three replicates of a single

experiment.

values greater than 20000 nm while keeping S1P₁potency sim-

ilar to that of compound 44.

For all compounds tested, only weak activity on S1P₄was

found (selectivity factor versus S1P₁ꢂ1000 fold).^[29]Most ana-

logues showed similar potency on S1P₁and S1P₅, except for

compounds 45 and 50, which showed selectivity factors of

1000 and 500, respectively. The high selectivity of compound

45 was unexpected taking into account the similarity of S1P₁

and S1P₅binding pocket residues.^[21b]

Exchange of the biphenyl substituents of 45 led to com-

pound 47 with improved affinities for both S1P₁as well as

S1P₃. However, the selectivity factor of greater than 2000 was

retained. Combining this same lipophilic part with previously

investigated head groups yielded compounds 48–50. These

compounds all demonstrated good S1P₁potency with K_i

values ranging from 0.48 to 0.98 nm and S1P₃selectivity factors

between 520 and 662 fold. Solid-state structures of both the

parent and the chloride salt of compound 49 have been

solved by single-crystal X-ray analyses, and the structures re-

vealed conformations consistent with expectations and the

pharmacophore hypothesis.^[28]The SAR study of these com-

pounds indicated clearly that each moiety has an impact on

potency, S1P₃selectivity, and overall physicochemical proper-

ties.

In vitro metabolism studies with liver microsomes indicated

that the metabolic clearance of the evaluated compounds is

low in human, rat and mouse (Table 5). CYP interaction by in-

hibition and induction was also evaluated, aiming to predict

the possibility of drug–drug interactions with other co-medi-

cated drugs. CYP inhibition by the seven selected compounds

was found to be weak, with IC₅₀values greater than 10 mm,

except for compounds 21, 26 and 48, which were consequent-

ly deprioritized (Table 5). CYP induction was measured for com-

pounds 45 and 49, and minor inhibitory activity was found for

compound 49 against CYP 2B at 100 mm. All compounds are

extensively bound to plasma proteins (>99.4% for human, rat

and mouse plasma), and low unbound fractions in brain

(<0.1%) were measured for compounds 45, 48 and 49.

In vitro Selectivity and Early ADMET Profile

From among the most potent H–P fragment combinations,

compounds 21, 26, 40, 45, 48, 49 and 50 were selected for

further characterization. We evaluated their selectivity versus

S1P_3ꢀ5isoforms in radioligand binding assays, early ADME fo-

cused on metabolism (intrinsic clearance), cytochrome P450

(CYP) interactions, unbound fraction, and early safety (Table 5).

Early safety and toxicity evaluation of this selection of ben-

zylic amine and ether derivatives was carried out. The com-

pounds were tested on hERG channel in an electrophysiology

assay (patch clamp assay), yielding K_ivalues greater than

10 mm, except for compounds 45 and 49 with K_ivalues of

4 mm and 9 mm, respectively. Considering the unbound fraction

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Table 5. Selectivity and in vitro ADME profiling of compounds 21, 26, 40, 45, 48, 49 and 50.

Compd 21

Compd 26

Compd 40

Compd 45

Compd 48

Compd 49

Compd 50

K_i[nm]^[a]

S1P₁

S1P₃

S1P₄

0.81ꢁ0.19

1040ꢁ85

N.D.

3.5ꢁ0.7

0.27ꢁ0.12

75.3ꢁ6.2

N.D.

2.50ꢁ0.55

0.20ꢁ0.07

22.2ꢁ0.4

381ꢁ270

12.9ꢁ7.3

1.09ꢁ0.38

>20000

5550ꢁ490

1020ꢁ250

0.71ꢁ0.21

470ꢁ160

895ꢁ290

36.0ꢁ9.9

0.48ꢁ0.21

251.6ꢁ93

605ꢁ210

8.8ꢁ4.6

0.98ꢁ0.14

603ꢁ24

> 20000

530ꢁ230

S1P₅

[b]

Cl_int[mL/minmg^ꢀ1

]

human

rat

mouse

<10

N.D.

29

18

N.D.

<10

12

N.D.

<10

18

<10

12

<10

N.D.

CYP Inh.^[c]

IC₅₀[mm]

2C19=6.7

2C8=9.3

3A4=7.3

>10

2C8=3.9

>10

Fraction unbound

plasma (human)

plasma (rat)

plasma (mouse)

brain (mouse)

N.D.

0.63

0.6

0.48

N.D.

0.21

0.14

0.21

N.D.

0.08

0.06

0.07

0.04

<0.05

0.08

0.06

0.11

0.19

0.14

0.02

<0.05

N.D.

0.02

hERG^[d]

Genotoxicity^[e]

13ꢁ5%

N.D.

ATꢀ

24ꢁ4%

MNTꢀ

4000 nm

ATꢀ

11300 nm

ATꢀ

9000 nm

ATꢀ

6ꢁ4%

MNTꢀ

N.D.

[a] K_iare the mean ꢁSD of three replicates and a minimum of two independent experiments. N.D.=not determined. [b] Intrinsic clearance (Cl_int) deter-

mined against human, rat and mouse microsomes. [c] Inhibition of cytochrome P450 (CYP) isoforms; values are >10 mm against all isoforms evaluated

unless specified otherwise. [d] hERG inhibition was determined using a patch clamp assay; data represent the K_ivalue [nm] or % inhibition at 10 mm.

[e] Genotoxicity was evaluated in vitro using a micronucleus (MNT) or Ames (AT) test; all compounds tested gave negative results, presented as MNTꢀ or

ATꢀ.

available in vivo, the cardiovascular risk associated with hERG

inhibition is estimated to be acceptable for all compounds.

Furthermore, when evaluated in vitro for genotoxicity using

Table 6. Lymphopenia and pharmacokinetic (PK) parameters after oral

(po) administration to mice.^[a]

a micronucleus or Ames test, all compounds were found to be

negative (Table 5).

Selectivity of compounds 45 and 49 was evaluated by

screening against a panel of receptors, ion channels, transport-

ers and kinases. No appreciable activities at concentrations

below 1 mm were identified, except for compound 45, which

exhibited binding activity on H₁and H₂receptors with a K_i

value of 500 nm.^[30]Overall, these pharma profiles suggest

a low potential for off target effects for these derivatives, in

spite of the frequent use of oxadiazole in drug discovery pro-

grams across diverse target classes and therapeutic areas.^[25]

Compd 40 Compd 45 Compd 49 Compd 50

Lymphopenia at 48 h^[b]

at 30 mgkg^ꢀ1

at 10 mgkg^ꢀ1

at 3 mgkg^ꢀ1

68ꢁ3

75ꢁ1

70ꢁ3

12ꢁ9

71ꢁ2

57ꢁ3

36ꢁ5

29ꢁ7

16ꢁ3

12ꢁ4

ꢀ1ꢁ8

3ꢁ7

[c]

PK parameters (30 mgkg^ꢀ1

)

AUC [h*ngmL^ꢀ1

]

202751

10293

4

265151

13267

2

106171

8530

2

227668

22433

2

1

C_max[ngmL^ꢀ1

]

t

_max[h]

t_1/2[h]

Cl/F [Lkg^ꢀ1h^ꢀ1

5.3

0.15

14.9

0.1

7.8

0.3

6.3

0.1

]

Exposure [ngmL^ꢀ1

Plasma at 24 h

Plasma at 48 h

LN at 24 h

LN at 48 h

Brain at 24 h

Brain at 48 h

]

PK/PD Lymphopenia and EAE Models

3823

123

3143

85

1504

37

5701

1576

6103

1309

16172

6944

1429

145

3414

386

4275

332

849

124

3765

501

4536

648

Decrease of peripheral lymphocyte count has frequently been

used as a PD biomarker for S1P₁agonist efficacy in autoim-

mune diseases such as MS. This model was thus employed to

further characterize the optimized compounds in vivo. De-

crease in circulating blood lymphocytes was measured at

48 hours after a single oral gavage, along with PK parameters.

This PK/PD experiment also provided data for the total drug

exposure over time as estimated by the area under the curve

[a] Test compound was administered orally to female C57BL/6 mice as

a suspension in 0.5% carboxymethylcellulose (CMC)/0.25% Tween20 in

water. [b] Values represent the percentage of the basal value; each treate-

ment group was comprised of n=8 animals; values are expressed as the

mean ꢁSEM. [c] PK parameters: total drug exposure in plasma over time

(AUC ); maximum plasma concentration (C_max); time taken to reach C_max

(AUC ), the maximum plasma concentration (C_max), the elimina-

1

(t_max); elimination half-life (t_1/2); clearance (Cl)/bioavailability (F) ratio; com-

pound exposure in plasma, lymph nodes (LN) and brain.

tion half-life (t_1/2), the clearance (Cl) to bioavailability (F) ratio,

and brain and lymph node (LN) exposure (Table 6).

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A dose of 30 mgkg^ꢀ1was initially selected for PK parameter

determination and lymphocyte counts at 48 hours (Table 6). A

long lasting lymphopenic effect was targeted in view of better

efficacy.^[31]At this dose, significant lymphopenia was observed

for all selected compounds in correlation with plasma expo-

sure. Higher distribution in LN and brain compared with

plasma was measured for all compounds with the exception of

40.

We recognized that some of the efficacy of fingolimod in MS

could be due to actions on S1P receptors in the CNS, therefore

the brain penetration of these compounds can be considered

an asset.^[4]Accumulation in lymphoid tissues was also reported

for fingolimod (Ia) and has been linked to its prolonged effica-

cy on the S1P₁receptor.^[31]This benchmark compound demon-

strated profound lymphopenia (Figure 4), due to its very spe-

cific, albeit well-described, PK profile.

Compound 49, an S1P_1,5agonist, and compound 45, an

S1P₁-selective agonist, were benchmarked head to head with

fingolimod (FTY-720, Ia) in an experimental autoimmune ence-

phalomyelitis (EAE) murine disease model.^[32]This model

mimics MS by inducing a demyelinating autoimmune response

by utilizing MOG_35–55as an autoantigen.^[33]Leukopenia and

plasma exposure were determined 24 hours after the last

dosing. S1P_1,5agonist 49 demonstrated efficacy comparable

with fingolimod at doses of 1 and 3 mgkg^ꢀ1for 3611 days (Fig-

ure 5D,E). The selective S1P₁agonist, compound 45, was less

efficacious than fingolimod even at 3 mgkg^ꢀ1and a treatment

period of 44 days (Figure 5A,B). Interestingly, leukopenia mea-

sured 24 hours after the last dosing was similar for both com-

pounds 45 and 49 (Figure 5C,F). As plasma exposures are con-

sistently higher for compound 45 versus compound 49, the su-

periority of compound 49 cannot be attributed to favourable

PK.^[34]This difference in efficacy in the MOG_35–55-induced EAE

model is revealing. The main distinction of compound 45 is its

lack of activity against S1P₅, whose involvement in rodent MS

models has been disputed.^[7]The head-to-head comparison of

analogues of the same series, with similar S1P₁potencies,

physicochemical and ADME properties but different S1P recep-

tor affinity profiles, opens the opportunity to address this

question in detail. In the case at hand, however, the presented

results led to the selection of compound 49 as a clinical candi-

date.

Candidates for the subsequent EAE disease model were se-

lected after lymphopenia evaluation at lower concentrations.

Only compounds 45 and 49 demonstrated efficacious lympho-

penia at 10 mgkg^ꢀ1and to a lower extent at 3 mgkg^ꢀ1(Table 6

and Figure 4); these two compounds also exhibited the lon-

gest half-life. These results make the compound pair 45 and

49, two very similar S1P₁agonists from the same chemical

series but with significantly different S1P₅activity, a very prom-

ising probe set for investigations aiming to dissect the contri-

butions of S1P₅to S1P₁-mediated biological effects.

Figure 4. Treatment with S1P receptor agonists induced lymphopenia in C57BL/6 mice. Mice received oral treatment with compound 40 (A), 45 (B), 49 (C) or

50 (D), and blood lymphocytes were counted 48 h later. Each treatment group was comprised of n=8 animals. Statistical analysis was performed by ANOVA

followed by Newman–Keuls multiple comparison test (* P<0.05; *** P<0.001). Values are expressed as the mean ꢁSEM.

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Figure 5. Treatment with compound 45 or 49 induced a therapeutic effect in the MOG_35–55-induced EAE model in C57BL/6 mice. Mice were immunized with

MOG_35–55and treated with 45 (A, B and C), 49 (D, E and F) or FTY-720 (A–F) at the indicated dose from day 14 post-immunization until study termination, as

described in the Experimental Section. The arrow indicates the day of the beginning of treatment. Each treatment group was comprised of n animals: vehicle

(n=12 in A, n=8 in D), FTY-720 at 1 mgkg^ꢀ1(n=12 in A, n=8 in D), 45 or 49 at 1 and 3 mgkg^ꢀ1(n=12 and 9, respectively), Sham (n=5). A and D) Data

were analyzed by the Kruskal–Wallis method followed by Dunn’s multiple comparisons test. The bars placed above the graph are in line with each treatment

group to indicate the period during which a statistically significant difference has been found in comparison with the vehicle-treated group as follows:

* P <0.05 for vehicle vs FTY-720 at 1 mgkg^ꢀ1; § P <0.05 for vehicle vs 45 or 49 at 1 mgkg^ꢀ1; # P <0.05 for vehicle vs 45 or 49 at 3 mgkg^ꢀ1. B and E) The clin-

ical efficacy was analyzed by measuring the area under the curve (AUC) of the clinical score from beginning of treatment until study termination. AUC data

were analyzed by the Kruskal–Wallis method followed by Dunn’s multiple comparisons test (* P <0.05; ** P <0.01 vs vehicle group). C and F) Upon termina-

tion of the experiment, 24 h after the last administration of compound, blood was harvested, and total leukocytes were counted for assessment of the leuko-

penia. Leukopenia data were analyzed by ANOVA followed by a Newman–Keuls multiple comparison test (** P <0.01; *** P <0.001). For B, C, E and F, data

for the inhibition (%) versus the vehicle group are shown above each bar. Values are expressed as mean ꢁSEM.

Conclusions

parable with the marketed agent, fingolimod, which led to its

selection as a clinical candidate. Compound 49 is not a pro-

drug, and its improved property profile, most notably its selec-

tivity, should translate into a safer treatment of relapsing forms

of multiple sclerosis.

This report summarizes the genesis of a series of novel oxadia-

zoles as selective S1P receptor agonists, its design and optimi-

zation supported by a pharmacophore model and calculated

physicochemical properties. An extensive SAR study of 48 de-

rivatives allowed the association of fragments and their combi-

nations with their impact on selectivity, early toxicology and Experimental Section

PK/PD. After close examination of the data, compounds 45

and 49 were identified as S1P₁- and S1P_1,5-selective frontrun-

Computational methods

ners, respectively. These compounds induced long-lasting de-

creases in lymphocyte count after oral administration and

showed preferential distribution in lymph nodes and brain. In

S1P₁pharmacophore: A Catalyst pharmacophore model

(Figure 6) was constructed using HipHop^[20]from nine diverse

S1P₁ligands from the literature and internal screening efforts.

The model contains three hydrophobic centers and two hydro-

gen-bond acceptors with their projected donor sites. In an

a

head-to-head comparison in an MOG_35–55-induced EAE

murine model, only compound 49 demonstrated efficacy com-

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Method E: column: Waters Xterra MS C₁₈(100ꢂ4.6 mm, 5 mm) plus

guard cartridge; flow rate: 2 mLmin^ꢀ1; mobile phase: 10 mm

NH₄HCO₃in H₂O (solvent A) and CH₃CN (solvent B); conditions (A/

B): 95:5!5:95 over 3.5 min, then held at 5:95 for 1.5 min.

Preparative HPLC: Purifications were performed with a mass-direct-

ed autopurification (MDAP) FractionLynx from Waters equipped

with a sunfire prep C₁₈OBD column (19ꢂ100 mm5 mm), unless

otherwise noted. All HPLC purifications were performed with a gra-

dient of H₂O (0.1% HCOOH)/CH₃CN (0.1% HCOOH) 95:5!0:100

over 15 to 20 min with a flow rate of 20 mLmin^ꢀ1

.

Figure 6. Geometrical arrangement of the pharmacophore; distances be-

tween features in ꢁ; colour coding as in Figure 3.

Mass spectrometry: Liquid chromatography (LC)/MS analyses

were performed on Waters Alliance 2795 coupled with

a

a ZSprayꢃ mass detector (ZMD) [electrospray (ES)] equipped with

a Waters X-Bridge column (C₈30ꢂ2.1 mm 3.5 mm). Ultra-high-per-

formance liquid chromatography (UHPLC)/MS analyses were per-

formed on a Waters Acquity coupled with Waters Acquity S70QD

(ES) equipped with a Waters Acquity BEH column (C₁₈50ꢂ2.1 mm

1.7 mm), using the following conditions: CH₃CN/H₂O (NH₄OAc

10 mm), 5 to 100% (2–3 min), max plot 230–400 nm.

a posteriori validation against 178 S1P receptor modulators

from a recent comprehensive Review article,^[26]the model was

able to retrieve up to 148 (83%) of the known actives.

Computed physical properties: Topological polar surface area

(TPSA), pK_aand cLogD values were calculated using ACD/Labs

PhysChem, version 12.01 (2009), from Advanced Chemistry

Development Inc., Toronto, Canada, www.acdlabs.com.

Methyl 4-[amino(hydroxyimino)methyl]-2-fluorobenzoate (52a):

Methyl 4-cyano-2-fluorobenzoate (17.76 g, 99.1 mmol) was sus-

pended in EtOH (200 mL). NH₂OH (50% in H₂O, 30 mL, 495 mmol)

was added, and the resulting yellow suspension was stirred at RT

overnight. The suspension was filtered, and the remaining residue

was rinsed with EtOH (2ꢂ50 mL) and dried in vacuo, affording 52a

Synthetic procedures

1

as a white solid (18.91 g, 90%): H NMR (300 MHz, [D₆]DMSO): d=

General: The commercially available starting materials used in the

10.09 (s, 1H), 7.95–7.89 (m, 1H), 7.70–7.62 (m, 2H), 6.05 (s, 2H),

3.89 ppm (s, 3H); UHPLC/MS: m/z: 210.9 [MꢀH]^ꢀ, 212.9 [M+H]⁺;

HPLC (method A): t_R=0.97 min (purity: 100%).

following experimental description were purchased from Aldrich,

1

Fluka, Acros or ABCR unless otherwise noted. H NMR spectra were

recorded on BRUKER spectrometers, models DPX-300, AV-II or AV-III

400 MHz. Chemical shifts (d) are reported in parts per million

(ppm) relative to the residual solvent peak; multiplicity is denoted

using standard abbreviations, and coupling constants (J) are re-

ported in Hertz (Hz). Microwave reactions were performed in

a single-mode microwave reactor Emrys Optimiser from Personal

Chemistry or a single-mode microwave reactor Initiator 60 from Bi-

otage in sealed reaction vessels. Elemental analyses were per-

formed on an Erba Science 11108 CHN analyzer.

tert-Butyl {4-[amino(hydroxyimino)methyl]-2-fluorophenoxy}ace-

tate

(52b):

Step 1:

3-Fluoro-4-hydroxybenzonitrile

(2 g,

14.59 mmol) was dissolved in CH₃CN (120 mL). CsCO₃(5.70 g,

17.50 mmol) and tert-butyl bromoacetate (2.26 mL, 15.32 mmol)

were added, and the mixture was stirred at RT for 12 h. The reac-

tion mixture was concentrated, and the crude mixture was diluted

with EtOAc (50 mL) and washed with water (3ꢂ30 mL) then with

brine (30 mL), dried over MgSO₄, filtered and concentrated, afford-

ing tert-butyl (4-cyano-2-fluorophenoxy)acetate as a yellow solid

HPLC: Analyses were performed on a Waters 2695 instrument

equipped with a Waters 996 photodiode array detector. Trifluoro-

acetic acid (TFA), formic acid (HCOOH), CH₃CN and H₂O were used

as eluents.

1

(3.58 g, 97%): H NMR (300 MHz, [D₆]DMSO): d=7.91–7.84 (m, 1H),

7.68–7.65 (m, 1H), 7.37–7.24 (m, 1H), 4.92 (s, 2H), 1.42 ppm (s, 9H);

UHPLC/MS: m/z: 250.2 [MꢀH]^ꢀ; HPLC (method A): t_R=4.72 min

(purity: 97.6%).

Method A: column: X-Bridge C₈(50ꢂ4.6 mm3.5 mm); flow rate:

2 mLmin^ꢀ1

; mobile phase: H₂O+0.1% TFA (solvent A) and

Step 2: Compound 52b was prepared following the procedure de-

scribed for 52a, starting from tert-butyl (4-cyano-2-fluorophenox-

y)acetate. It was isolated as a white solid (3.57 g, 90%): ¹H NMR

(300 MHz, [D₆]DMSO): d=9.61 (brs, 1H), 7.51–7.41 (m, 2H), 7.09–

7.03 (t, J=8.84 Hz, 1H), 5.81 (brs, 2H), 4.78 (s, 2H), 1.42 ppm (s,

9H); UHPLC/MS: m/z: 285.1 [M+H]⁺; HPLC (method A): t_R=

2.91 min (purity: 94.3%).

CH₃CN+0.1% TFA (solvent B); conditions (A/B): 95:5!5:95 over

8 mins.

Method B: column: ATLANTIS C₁₈(50ꢂ4.6 mm, 5 mm); flow rate:

1 mLmin^ꢀ1; mobile phase: H₂O+0.1% HCOOH (solvent A) and

CH₃CN (solvent B); conditions (A/B): 90:10!0:100 over 4 min, then

held at 100% solvent B for 2 mins.

tert-Butyl ({4-[(Z)-amino(hydroxyimino)methyl]-2-fluorobenzyl}-

oxy)acetate (52c): Step 1: A solution of NaOH (20 g) in H₂O

(40 mL) was added to a mixture of 2-tert-butyl glycolate (1.38 g,

10.47 mmol) and tetrabutylammonium hydrogen sulfate (317 mg,

0.93 mmol) in toluene (40 mL). 4-Cyano-2-fluorobenzylbromide

(2.00 g, 9.34 mmol) was added, and the resulting mixture was

stirred at RT overnight. The layers were separated. The aqueous

layer was extracted with EtOAc (2ꢂ30 mL). The organic layers were

combined, washed with saturated aq NH₄Cl (30 mL) and brine

(30 mL), dried over MgSO₄, filtered and concentrated in vacuo.

After purification by flash chromatography (EtOAc/heptane,

Method C: column: Phenomenex Luna C₁₈(2) (100ꢂ4.6 mm, 5 mm)

plus guard cartridge; flow rate: 2 mLmin^ꢀ1; mobile phase: H₂O+

0.1% HCOOH (solvent A) and CH₃CN+0.1% HCOOH (solvent B);

conditions (A/B): 95:5!5:95 over 3.5 min, then held at 5:95 for

2 min.

Method D: column: Waters Xbridge C₁₈(2) (250ꢂ4.6 mm, 5 mm)

plus guard cartridge; flow rate: 1 mLmin^ꢀ1; mobile phase: 10 mm

NH₄HCO₃in H₂O (solvent A) and CH₃CN (solvent B); conditions (A/

B): 95:5!0:100 over 19.5 min, then held at 100% solvent B for

4 min.

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10:90!60:40), tert-butyl [(4-cyano-2-fluorobenzyl)oxy]acetate was

obtained as a colorless oil (1.58 g, 64%): ¹H NMR (300 MHz,

[D₆]DMSO): d=7.87 (dd, J=9.9, 1.4 Hz, 1H), 7.74 (dd, J=7.9,

1.5 Hz, 1H), 7.71–7.64 (m, 1H), 4.68 (s, 2H), 4.12 (s, 2H), 1.43 ppm

(s, 9H); UHPLC/MS: m/z: 283.3 [M+NH₄]⁺; HPLC (method A): t_R=

4.38 min (purity: 99.9%).

with EtOAc (2ꢂ25 mL). The combined organic layers were washed

with water (25 mL), dried over MgSO₄, filtered and the solvent was

removed in vacuo to give tert-butyl N-(4-cyanobenzyl)-N-methylgly-

1

cinate as a yellow oil (1942 g, 98%): H NMR (300 MHz, [D₆]DMSO):

d=7.79 (d, J=8.4 Hz, 2H), 7.51 (d, J=8.4 Hz, 2H), 3.71 (s, 2H), 3.20

(s, 2H), 2.23 (s, 3H), 1.42 ppm (s, 9H); UHPLC/MS: m/z: 260.9

[M+H]⁺; HPLC (method A): t_R=2.27 min (purity: 88%).

Step 2: Compound 52c was prepared following the procedure de-

scribed for 52a, starting from tert-butyl [(4-cyano-2-fluorobenzyl)-

oxy]acetate. It was isolated as a colorless oil (758 mg, 96%):

¹H NMR (300 MHz, [D₆]DMSO): d=9.79 (s, 1H), 7.57–7.39 (m, 3H),

5.90 (s, 2H), 4.59 (s, 2H), 4.05 (s, 2H), 1.43 ppm (s, 9H); UHPLC/MS:

m/z: 299.1 [M+H]⁺; HPLC (method A): t_R=2.92 min (purity: 97.7%).

Step 2: Compound 52 f was prepared following the procedure de-

scribed for 52a, starting from tert-butyl N-(4-cyanobenzyl)-N-meth-

ylglycinate, which was used as prepared in step 1 without further

purification. Compound 52 f was isolated as a white powder

1

(12.54 g, 95%): H NMR (300 MHz, [D₆]DMSO): d=9.58 (s, 1H), 7.62

(d, J=8.3 Hz, 2H), 7.28 (d, J=8.3 Hz, 2H), 5.78 (s, 2H), 3.62 (s, 2H),

3.15 (s, 2H), 2.24 (s, 3H), 1.42 ppm (s, 9H); UHPLC/MS: m/z: 294.0

[M+H]⁺; HPLC (method A): t_R=1.33 min (purity: 90%).

tert-Butyl

({3-[amino(hydroxyimino)methyl]benzyl}oxy)acetate

(52d): Compound 52d was prepared following the procedure de-

scribed for 52c, starting from 3-cyanobenzyl alcohol (3 g,

22.53 mmol) and tert-butyl bromoacetate (3.73 mL, 25.23 mmol). It

was isolated as a white powder (4.48 g, 91%): ¹H NMR (300 MHz,

[D₆]DMSO): d=9.62 (s, 1H), 7.65 (brs, 1H), 7.63–7.54 (m, 1H), 7.41–

7.31 (m, 2H), 5.80 (s, 2H), 4.54 (s, 2H), 4.04 (s, 2H), 1.43 ppm (s,

9H); UHPLC/MS: m/z: 279.2 [MꢀH]^ꢀ, 281.2 [M+H]⁺; HPLC (meth-

od A): t_R=2.75 min (purity: 100%).

tert-Butyl N-{4-[amino(hydroxyimino)methyl]benzyl}-N-methyl-b-

alaninate (52g): Compound 52g was prepared following the pro-

cedure described for 52 f, starting from 4-cyanobenzyl bromide

and tert-butyl N-methyl-b-alaninate.^[30]It was isolated as a yellow

oil (1.6 g, 77%): ¹H NMR (300 MHz, [D₆]DMSO): d=9.57 (brs, 1H),

7.61 (d, J=8.2 Hz, 2H), 7.27 (d, J=8.2 Hz, 2H), 5.77 (s, 2H), 3.51–

3.45 (m, 2H), 2.59 (t, J=7.0 Hz, 2H), 2.38 (t, J=7.0 Hz, 2H), 2.10 (s,

3H), 1.40 ppm (s, 9H); UHPLC/MS: m/z: 308.0 [M+H]⁺; HPLC

(method A): t_R=1.52 min (purity: 82.3%).

tert-Butyl N-{4-[amino(hydroxyimino)methyl]benzyl}-N-(tert-bu-

toxycarbonyl)glycinate (52e): Step 1: To a solution of 4-cyanoben-

zyl bromide (1.50 g, 7.65 mmol) in CH₃CN (25 mL) was added K₂CO₃

(2.11 g, 15.3 mmol) and tert-butyl glycinate (1.20 g, 9.18 mmol). The

reaction mixture was stirred at RT overnight. The solvent was re-

moved in vacuo, and the resulting mixture was diluted with water

(20 mL), extracted with EtOAc (2ꢂ25 mL), washed with brine

(30 mL), dried over MgSO₄, filtered and concentrated in vacuo. The

crude product was purified by flash chromatography (cyclohexane/

EtOAc, 90:10!40:60), affording tert-butyl N-(4-cyanobenzyl)glyci-

nate as colorless oil (1.14 g, 60%): ¹H NMR (300 MHz, [D₆]DMSO):

d=7.78 (d, J=8.3 Hz, 2H), 7.52 (d, J=8.3 Hz, 2H), 3.79 (s, 2H), 3.19

(s, 2H), 2.60 (brs, 1H), 1.41 ppm (s, 9H); UHPLC/MS: m/z: 247.0

[M+H]⁺; HPLC (method A): t_R=2.17 min (purity: 97.2%).

{tert-Butoxycarbonyl-[3-(N-hydroxycarbamimidoyl)benzyl]ami-

no}acetic acid tert-butyl ester (52h): Step 1: A solution of glycine-

tert-butyl ester (5 g, 38 mmol), 3-cyanobenzaldehyde (5 g,

0.0381 mol) in toluene (100 mL) was heated at reflux under N₂for

2 h. After evaporation of the solvent, the reaction mass was redis-

solved in dry MeOH (100 mL). NaBH₄(2.17 g, 57.1 mmol) was

added in portions at 08C, and the resulting mixture was stirred at

RT for 12 h. Water was added (100 mL), and the aqueous layer was

extracted with CH₂Cl₂(2ꢂ100 mL), dried over Na₂SO₄, filtered and

concentrated in vacuo to afford tert-butyl [(3-cyanobenzyl)amino]a-

cetate as a yellow oil (8.1 g, 86%): ¹H NMR (400 MHz, [D₆]DMSO):

d=7.71–7.72 (m, 2H), 7.63–7.65 (m, 1H), 7.53–7.55 (m, 1H), 5.40–

5.41 (m, 1H), 4.53–4.54 (m, 2H), 3.17 (s, 2H), 1.39 ppm (s, 9H).

Step 2: A mixture of tert-butyl N-(4-cyanobenzyl)glycinate (1.1 g,

4.5 mmol), di-tert-butyl dicarbonate (1.1 g, 4.9 mmol) and DIEA

(1.1 mL, 6.7 mmol) was prepared in CH₂Cl₂(22 mL) and stirred at

RT for 3.5 h. The reaction mixture was diluted with CH₂Cl₂(30 mL)

and washed with saturated aq NaHCO₃(2ꢂ20 mL) and brine

(20 mL). The organic layer was dried over MgSO₄, filtered and con-

centrated to afford tert-butyl N-(tert-butoxycarbonyl)-N-(4-cyano-

Step 2: Compound 52h was prepared following the procedure de-

scribed for 52e, starting from tert-butyl [(3-cyanobenzyl)amino]ace-

tate. It was isolated as a pale yellow oil (3.8 g, 99%): ¹H NMR

(400 MHz, [D₆]DMSO): d=9.59 (s, 1H), 7.53–7.56 (m, 2H), 7.29–7.33

(m, 1H), 7.24–7.26 (m, 1H), 5.76 (s, 2H), 4.37–4.39 (s, 2H), 3.74 (s,

2H), 1.35–1.37 ppm (s, 18H); UHPLC/MS: m/z: 380.0 [M+H]⁺; HPLC

(method B): t_R=5.91 min (purity: 90.3%).

1

benzyl)glycinate as a colorless oil (1.2 g, 77%): H NMR (300 MHz,

[D₆]DMSO): d=7.84–7.78 (m, 2H), 7.48 (d, J=8.0 Hz, 2H), 4.50–4.45

(m, 2H), 3.93–3.83 (m, 2H), 1.41–1.29 ppm (m, 18H); UHPLC/MS:

m/z: 347.1 [M+H]⁺; HPLC (method A): t_R=5.10 min (purity:

100.0%).

tert-Butyl [{3-[amino(hydroxyimino)methyl]benzyl}(methyl)ami-

no]acetate (52i): Compound 52i was prepared following the pro-

cedure described for 52 f, starting from 3-(bromomethyl)benzoni-

trile. It was isolated as a white powder (8.5 g, 84%): ¹H NMR

(400 MHz, [D₆]DMSO): d=9.57 (s, 1H), 7.59 (s, 1H), 7.54–7.51 (m,

1H), 7.31–7.28 (m, 2H), 5.75 (s, 2H), 3.61 (s, 2H), 3.15 (s, 2H), 2.23

(s, 3H), 1.41 ppm (s, 9H); UHPLC/MS: m/z: 294.0 [M+H]⁺; HPLC

(method A): t_R=3.31 min (purity: 97.5%).

Step 3: Compound 52e was prepared following the procedure de-

scribed for 52a, starting from tert-butyl N-(tert-butoxycarbonyl)-N-

(4-cyanobenzyl)glycinate. It was isolated as a colorless oil (1.26 g,

1

97%): H NMR (300 MHz, [D₆]DMSO): d=9.59 (s, 1H), 7.65–7.60 (m,

2H), 7.26 (d, J=8.3 Hz, 2H), 5.77 (s, 2H), 4.40 (s, 2H), 3.84–3.75 (m,

2H), 1.41–1.34 ppm (m, 18H); UHPLC/MS: m/z: 380.1 [M+H]⁺;

HPLC (method A): t_R=3.31 min (purity: 98.1%).

tert-Butyl

3-[{3-[amino(hydroxyimino)methyl]benzyl}(methyl)-

tert-Butyl N-{4-[amino(hydroxyimino)methyl]benzyl}-N-methyl-

glycinate (52 f): Step 1: A mixture of 4-(bromomethyl)benzonitrile

(1.50 g, 7.65 mmol), sarcosine tert-butyl ester hydrochloride (1.67 g,

9.18 mmol) and K₂CO₃(3.17 g, 22.95 mmol) in CH₃CN (25 mL) was

stirred at RT overnight. The reaction mixture was concentrated in

vacuo. The residue was diluted with water (25 mL) and extracted

amino]propanoate (52j): Compound 52j was prepared following

the procedure described for 52 f, starting from 3-(bromomethyl)-

benzonitrile and tert-butyl N-methyl-b-alaninate.^[30]It was isolated

as

a

white gummy solid (4.5 g, 84%): ¹H NMR (400 MHz,

[D₆]DMSO): d=9.56 (s, 1H), 7.51–7.53 (m, 2H), 7.27–7.31 (m, 2H),

5.74 (s, 2H), 3.44 (s, 2H), 2.55–2.59 (m, 2H), 2.34–2.38 (m, 2H), 2.09

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(s, 3H), 1.37 ppm (s, 9H); UHPLC/MS: m/z: 308.2 [M+H]⁺; HPLC

(method B): t_R=5.18 min (purity: 96.5%).

white solid (0.233 g, 78%): ¹H NMR (400 MHz, CDCl₃): d=7.72 (s,

1H), 7.63–7.52 (m, 2H), 4.84 (s, 2H), 3.76 (s, 2H), 3.19 (s, 2H), 2.37

(s, 3H), 1.48 ppm (s, 9H); LC/MS: m/z: 362 [M+H]⁺; HPLC (meth-

od E): t_R=2.36 min (purity: 93.24%)

tert-Butyl N-{2-[(Z)-amino(hydroxyimino)methyl]benzyl}-N-meth-

ylglycinate (52k): Compound 52k was prepared following the

procedure described for 52 f, starting from 2-(bromomethyl)benzo-

nitrile. It was isolated as a white solid (3.47 g, 81%): ¹H NMR

(300 MHz, [D₆]DMSO): d=9.39 (s, 1H), 7.45–7.24 (m, 4H), 6.07 (s,

2H), 3.67 (s, 2H), 3.16 (s, 2H), 2.25 (s, 3H), 1.40 ppm (s, 9H);

UHPLC/MS: m/z: 294.0 [M+H]⁺; HPLC (method A): t_R=1.99 min

(purity: 90.0%).

tert-Butyl 2-(((5-(N’-hydroxycarbamimidoyl)pyridin-2-yl)methyl)-

(methyl)amino)acetate (52p): Compound 52p was prepared fol-

lowing the procedure described for 52 f, starting from 6-bromome-

thylnicotinonitrile. It was isolated as an off-white solid (1.87 g,

77%): ¹H NMR (400 MHz, [D₆]DMSO): d=9.81 (s, 1H), 8.79 (d, J=

2 Hz, 1H), 8.03 (dd, J=8.2, 2.2 Hz, 1H), 7.48 (d, J=8.2 Hz, 1H), 5.97

(brs, 2H), 3.80 (s, 2H), 2.32 (s, 3H), 1.46 ppm (s, 9H).

tert-Butyl N-{4-[amino(hydroxyimino)methyl]-2-fluorobenzyl}-N-

methylglycinate (52l): Compound 52l was prepared following the

procedure described for 52 f, starting from 4-cyano-2-fluorobenzyl

bromide. It was isolated as white powder (323 g, 89%): ¹H NMR

(300 MHz, [D₆]DMSO): d=9.74 (s, 1H), 7.51 (dd, J=8.0, 1.6 Hz, 1H),

7.45–7.36 (m, 2H), 5.87 (s, 2H), 3.70 (s, 2H), 3.17 (s, 2H), 2.26 (s,

3H), 1.42 ppm (s, 9H); UHPLC/MS: m/z: 312.3 [M+H]⁺; HPLC

(method A): t_R=1.36 min (purity: 97.3%).

tert-Butyl N-{2-fluoro-3-[(hydroxyamino)(imino)methyl]benzyl}-N-

methylglycinate (52q): Compound 52q was prepared following

the procedure described for 52n, starting from 2-fluoro-3-methyl-

benzonitrile. It was isolated as a yellow oil (1 g, 85%): ¹H NMR

(300 MHz, [D₆]DMSO): d=9.57 (s, 1H), 7.49–7.30 (m, 2H), 7.22–7.09

(m, 1H), 5.77 (s, 2H), 3.70 (d, J=1.5 Hz, 2H), 3.18 (s, 2H), 2.26 (s,

3H), 1.42 ppm (s, 9H); UHPLC/MS: m/z: 312.0 [M+H]⁺; HPLC

(method A): t_R=1.09 min (purity: 92%).

2-Fluoro-N-hydroxy-4-hydroxymethyl-benzamidine (52m): Com-

pound 52m was prepared following the procedure described for

52a, starting from 2-fluoro-4-(hydroxymethyl)benzonitrile. It was

isolated as a white powder (12.54 g, 95%): ¹H NMR (400 MHz,

[D₆]DMSO): d=9.62 (s, 1H), 7.48 (t, J=7.7 Hz, 1H), 7.18 (d, J=

3.7 Hz, 1H), 7.16 (s, 1H), 5.79 (s, 2H), 5.42–5.35 (m, 1H), 4.55 ppm

(d, J=5.8 Hz, 2H); LC/MS: m/z: 185 [M+H]⁺; HPLC (method D): t_R=

8.16 min (purity: 97.0%).

tert-Butyl N-{5-[amino(hydroxyimino)methyl]-2-fluorobenzyl}-N-

methylglycinate (52r): Compound 52r was prepared following

the procedure described for 52 f, starting from 5-cyano-2-fluoro-

benzyl bromide. It was isolated as a colorless oil (602 mg, quantita-

1

tive): H NMR (300 MHz, [D₆]DMSO): d=9.61 (brs, 1H), 7.73 (dd, J=

7.3, 2.3 Hz, 1H), 7.62–7.56 (m, 1H), 7.20–7.12 (m, 1H), 5.81 (s, 2H),

3.71 (s, 2H), 3.20 (s, 2H), 2.28 (s, 3H), 1.43 ppm (s, 9H); UHPLC/MS:

m/z: 310.1 [MꢀH]^ꢀ, 312.2 [M+H]⁺; HPLC (method A): t_R=1.52 min

(purity: 94%).

tert-Butyl 2-((4-(N’-hydroxycarbamimidoyl)-2-(trifluoromethyl)-

benzyl)(methyl)amino)acetate (52n): Step 1: A solution of 4-

methyl-3-(trifluoromethyl)benzonitrile (1.85 g, 10 mmol), N-bromo-

succinimide (2.14 g, 12.0 mmol) and a,a’-azoisobutyronitrile (AIBN)

(0.033 g, 0.2 mmol) in CH₃CN (40 mL) was heated at reflux for 18 h.

The reaction mixture was diluted with EtOAc (40 mL) and water

(25 mL). The resulting suspension was filtered, and the filtrate was

separated. The organic phase was washed with brine (25 mL),

dried over MgSO₄, filtered and concentrated in vacuo. The residue

was purified by flash chromatography (iso-hexane/EtOAc, 90:10!

40:60). The resulting product was mixed with sarcosine tert-butyl

ester hydrochloride (0.516 g, 2.84 mmol) and K₂CO₃(0.980 g,

7.1 mmol) in CH₃CN (10 mL). The mixture was heated at 708C for

18 h. The solvent was evaporated in vacuo. The residue was dis-

solved in CH₂Cl₂(20 mL), washed with brine (15 mL), dried over

MgSO₄and filtered. The solvent was evaporated in vacuo, and the

resulting product was purified by flash chromatography (iso-

hexane/EtOAc, 90:10!40:60) to afford tert-butyl 2-((4-cyano-2-(tri-

tert-Butyl N-{3-[amino(hydroxyimino)methyl]-5-fluorobenzyl}-N-

methylglycinate (52s): Compound 52s was prepared following

the procedure described for 52n, starting from 3-fluoro-5-methyl-

benzonitrile. It was isolated as a yellow solid (850 mg, 75%):

¹H NMR (400 MHz, [D₆]DMSO): d=9.75 (brs, 1H), 7.49 (s, 1H), 7.34

(m, 1H), 7.13 (m, 1H), 5.87 (brs, 2H), 3.65 (s, 2H), 3.19 (s, 2H), 2.25

(s, 3H), 1.43 ppm (s, 9H); UHPLC/MS: m/z: 311.9 [M+H]⁺, 310.0

[MꢀH]^ꢀ; HPLC (method A): t_R=1.89 min (purity: 53%).

tert-Butyl N-{3-[amino(hydroxyimino)methyl]-6-fluorobenzyl}-N-

methylglycinate (52t): Compound 52t was prepared following

the procedure described for 52n, starting from 2-fluoro-5-methyl-

benzonitrile. It was isolated as a colorless oil (870 mg, 78%):

¹H NMR (300 MHz, [D₆]DMSO): d=9.59 (s, 1H), 7.42 (dd, J=7.1,

2.3 Hz, 1H), 7.37–7.25 (m, 1H), 7.17 (dd, J=10.6, 8.4 Hz, 1H), 5.78

(s, 2H), 3.59 (s, 2H), 3.15 (s, 2H), 2.22 (s, 3H), 1.41 ppm (s, 9H);

UHPLC/MS: m/z: 312.1 [M+H]⁺; HPLC (method A): t_R=1.23 min

(purity: 70%).

1

fluoromethyl)benzyl)(methyl)amino)acetate (0.543 g, 70%): H NMR

(400 MHz, CDCl₃): d=8.15 (d, J=8.2 Hz, 1H), 7.90 (s, 1H), 7.83 (d,

J=8.2 Hz, 1H), 3.90 (s, 2H), 3.26 (s, 2H), 2.37 (s, 3H), 1.48 ppm (s,

9H).

tert-Butyl N-[3-[amino(hydroxyimino)methyl]-5-(trifluoromethyl)-

benzyl]-N-methylglycinate (52u): Compound 52u was prepared

following the procedure described for 52n, starting from 3-methyl-

5-(trifluoromethyl)benzonitrile. It was isolated as a colorless oil

(790.0 mg, 83%): ¹H NMR (300 MHz, [D₆]DMSO): d=9.82 (s, 1H),

7.95–7.83 (m, 2H), 7.70–7.59 (m, 1H), 5.98 (s, 2H), 3.72 (s, 2H), 3.20

(s, 2H), 2.25 (s, 3H), 1.41 ppm (s, 9H); UHPLC/MS: m/z: 362.2

[M+H]⁺, 360.2 [MꢀH]^ꢀ; HPLC (method A): t_R=1.90 min (purity:

95.5%).

Step 2: Compound 52n was prepared following the procedure de-

scribed for 52a, starting from tert-butyl 2-((4-cyano-2-(trifluorome-

thyl)benzyl)(methyl)amino)acetate. It was isolated as white solid

(0.460 g, 77%): ¹H NMR (400 MHz, CDCl₃): d=7.97 (d, J=8.1 Hz,

1H), 7.89 (s, 1H), 7.78 (d, J=8.1 Hz, 1H), 4.86 (brs, 2H), 3.86 (s,

2H), 3.23 (s, 2H), 2.37 (s, 3H), 1.48 ppm (s, 9H); LC/MS: m/z: 362

[M+H]⁺, 360 [MꢀH]^ꢀ; HPLC (method E): t_R=3.38 min (purity:

84.8%).

2,2’-Dimethyl-1,1’-biphenyl-4-carboxylic acid (56d): Step 1: To

a solution of methyl 4-bromo-3-methylbenzoate (ABCR) (15 g,

65 mmol) in toluene (200 mL) and H₂O (200 mL), was added o-tolyl-

boronic acid (10.68 g, 78 mmol) followed by K₂CO₃(45.25 g,

32.7 mmol) and Pd(PPh₃)₄(3.78 g, 3.3 mmol). The mixture was de-

gassed with N₂and then heated at reflux (1208C) for 6 h. After

tert-Butyl 2-((4-(N’-hydroxycarbamimidoyl)-3-(trifluoromethyl)-

benzyl)(methyl)amino)acetate (52o): Compound 52o was pre-

pared following the procedure described for 52n, starting from 4-

methyl-2-(trifluoromethyl)benzonitrile. It was isolated as an off-

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completion, the reaction mixture was cooled to RT. The organic

phase was separated and evaporated in vacuo. The crude com-

pound was passed through a silica gel (60–120 mesh) column

using hexane as an eluent to obtain methyl 2,2’-dimethyl-1,1’-bi-

over MgSO₄, and filtered. After evaporation of the solvent, a yellow

solid was isolated. It was washed with pentane (2ꢂ500 mL), afford-

ing methyl 4-bromo-3-(bromomethyl)benzoate as a yellow solid

(42 g, 62%): H NMR (300 MHz, [D₆]DMSO): d=8.24 (d, J=1.91 Hz,

1H), 7.88–7.82 (m, 2H), 4.87 (s, 2H), 3.91 ppm (s, 3H); HPLC (meth-

1

phenyl-4-carboxylate as

a

white solid (15 g, 95%): ¹H NMR

(400 MHz, [D₆]DMSO): d=7.91 (s, 1H), 7.83–7.81 (m, 1H), 7.33–7.30

(m, 2H), 7.28–7.26 (m, 1H), 7.25–7.22 (m, 1H), 7.07–7.05 (m, 1H),

3.84 (s, 3H), 2.04 (s, 3H), 1.97 ppm (s, 3H); LC/MS: m/z: 240.9

[M+H]⁺; HPLC (method A): t_R=3.01 min (purity: 98.71%).

od A): t_R=4.44 min (purity: 97.9%).

Step 2: A solution of methyl 4-bromo-3-(bromomethyl)benzoate

(37.50 g, 121.77 mmol) in MeOH (1.125 L) was heated at reflux for

4 days. The reaction was concentrated and then was partitioned

between EtOAc (500 mL) and water (200 mL). The organic layer

was washed with 5% aq NaHCO₃(200 mL), brine (200 mL), dried

over MgSO₄, and filtered. After evaporation of the solvent, methyl

4-bromo-3-(methoxymethyl)benzoate was isolated as a beige solid

Step 2: To a solution of methyl 2,2’-dimethyl-1,1’-biphenyl-4-carbox-

ylate (15 g, 62.2 mmol) in THF (100 mL) was added 10% aq NaOH

(100 mL), and the mixture was heated at 1008C overnight. THF was

removed in vacuo, and the aqueous residue was washed with

EtOAc (100 mL). The aqueous layer was then acidified with 3n HCl

to pH 2–3 and extracted with CH₂Cl₂(2ꢂ100 mL). The combined

organic phase was washed with water (100 mL), dried over Na₂SO₄,

filtered and concentrated in vacuo to obtain 56d as a white solid

(13.5 g, 95%): ¹H NMR (400 MHz, [D₆]DMSO): d=12.89 (br s, 1H),

7.89 (s, 1H), 7.82–7.80 (m, 1H), 7.32–7.23 (m, 3H), 7.19–7.11 (m,

1H), 7.07–7.05 (m, 1H), 2.04 (s, 3H), 1.98 ppm (s, 3H); LC/MS: m/z:

227.0 [M+H]⁺; HPLC (method B): t_R=4.1 min (purity: 99.6%).

1

(29.8 g, 94%): H NMR (300 MHz, [D₆]DMSO): d=8.06–8.05 (m, 1H),

7.83 (d, J=1.23 Hz, 2H), 4.54–4.51 (m, 2H), 3.90 (s, 3H), 3.45 ppm

(s, 3H); UHPLC/MS: m/z: 227.2 [MꢀH]^ꢀ; HPLC (method A): t_R=

4.42 min (purity: 93.0%).

Step 3: A mixture of methyl 4-bromo-3-(methoxymethyl)benzoate

(40 g, 154.38 mmol), o-tolylboronic acid (23.09 g, 169.82 mmol),

K₂CO₃(106.68 g, 771.90 mmol) and Pd(PPh₃)₄(1.78 g, 1.54 mmol)

was dissolved in toluene (200 mL) and water (200 mL) under inert

atmosphere. The reaction mixture was purged with vacuum, de-

gassed with N₂, and then heated at reflux for 1 h. The reaction mix-

ture was cooled to RT, filtered over a pad of celite and washed

with EtOAc (1 L). The filtrate was concentrated to afford a yellow

oil, which was taken up in EtOAc (800 mL). The organic layer was

washed with saturated aq NaHCO₃(250 mL), water (250 mL) and

brine (250 mL), dried over MgSO₄, filtered and concentrated afford-

ing methyl 2-(methoxymethyl)-2’-methylbiphenyl-4-carboxylate as

a yellow oil (41.9 g, quantitative): ¹H NMR (300 MHz, [D₆]DMSO):

d=8.11 (dq, J=1.9, 0.6 Hz, 1H), 7.93 (ddd, J=7.8, 2.2, 0.6 Hz, 1H),

7.38–7.21 (m, 4H), 7.09 (dt, J=7.0, 1.1 Hz, 1H), 4.21–4.04 (m, 2H),

3.89 (s, 3H), 3.19 (s, 3H), 2.04–1.92 ppm (m, 3H); UHPLC/MS: m/z:

271.2 [M+H]⁺; HPLC (method A): t_R=5.34 min (purity: 89.4%).

2-Methoxy-2’-methylbiphenyl-4-carboxylic acid (56e): Com-

pound 56e was prepared following the procedure described for

56d, starting from methyl 4-bromo-3-methoxybenzoate. It was iso-

1

lated as a beige solid (1.95 g, 83%): H NMR (300 MHz, [D₆]DMSO):

d=13.06 (s, 1H), 7.66–7.61 (m, 2H), 7.31–7.23 (m, 4H), 7.14 (d, J=

7.28 Hz, 1H), 3.80 (s, 3H), 2.07 ppm (s, 3H); UHPLC/MS: m/z: 240.9

[MꢀH]^ꢀ; HPLC (method A): t_R=4.05 min (purity: 97.3%).

2’-Methyl-2-(trifluoromethyl) biphenyl-4-carboxylic acid (56 f):

Compound 56 f was prepared following the procedure described

for 56d, starting from methyl 4-bromo-3-(trifluoromethyl)benzoate.

1

It was isolated as off-white solid (4.07 g, 94%): H NMR (300 MHz,

[D₆]DMSO): d=13.55 (brs, 1H), 8.31 (s, 1H), 8.25 (d, J=7.90 Hz,

1H), 7.50 (d, J=7.90 Hz 1H), 7.37–7.12 (m, 4H), 1.99 ppm (s, 3H);

UHPLC/MS: m/z: 278.9 [MꢀH]^ꢀ; HPLC (method A): t_R=4.57 min

(purity: 98.7%).

Step 4:A solution of methyl 2-(methoxymethyl)-2’-methylbiphenyl-

4-carboxylate (40 g, 147.97 mmol), which was used as isolated in

step 3 without further purification, in EtOH (1.2 L) at RT was treated

with 5m aq NaOH (88.78 mL, 443.90 mmol). The reaction mixture

was stirred at 608C for 1 h. It was cooled to RT and concentrated

to give a yellow solid, which was dissolved in water (800 mL), and

the aqueous phase was washed with EtOAc (2ꢂ400 mL). The aque-

ous phase was acidified with concentrated HCl (40 mL) to pH 2

and then extracted with EtOAc (2ꢂ400 mL). The combined organic

extracts were washed with brine (2ꢂ400 mL), dried over MgSO₄, fil-

tered and concentrated affording compound 56a as a yellow solid

(35.1 g, 92%): ¹H NMR (300 MHz, [D₆]DMSO): d=12.99 (brs, 1H),

8.09 (s, 1H), 7.92–7.89 (m, 1H), 7.33–7.22 (m, 4H), 7.10–7.08 (m,

1H), 4.11 (m, 2H), 3.18 (s, 3H), 1.99 ppm (s, 3H); UHPLC/MS: m/z:

255.2 [MꢀH]^ꢀ; HPLC (method A): t_R=4.52 min (purity: 96.4%).

2’-Methoxy-2-methylbiphenyl-4-carboxylic acid (56g): Com-

pound 56g was prepared following the procedure described for

56d, starting from 2-methoxyphenylboronic acid. It was isolated as

a brown solid (1.12 g, 59%): ¹H NMR (300 MHz, [D₆]DMSO): d=

12.89 (s, 1H), 7.86 (s, 1H), 7.81 (dd, J=7.99, 1.23 Hz, 1H), 7.46–7.40

(m, 1H), 7.26–7.23 (d, J=7.68 Hz, 1H), 7.16–7.04 (m, 3H), 3.75 (s,

3H), 2.13 ppm (s, 3H); UHPLC/MS: m/z: 240.9 [MꢀH]^ꢀ; HPLC

(method A): t_R=4.05 min (purity: 98.5%).

2-Methyl-2’-(trifluoromethyl)biphenyl-4-carboxylic acid (56h):

Compound 56h was prepared following the procedure described

for 56d, starting from 2-(trifluoromethyl)benzeneboronic acid. It

was isolated as a white powder (22.5 g, 92%): ¹H NMR (300 MHz,

[D₆]DMSO): d=13.00 (s, 1H), 7.87 (m, 2H), 7.80 (dd, J=7.9, 1.6 Hz,

1H), 7.75–7.71 (m, 1H), 7.64–7.61 (m, 1H), 7.34 (d, J=7.6 Hz, 1H),

7.23 (d, J=7.9 Hz, 1H), 2.02 ppm (s, 3H); UHPLC/MS: m/z: 279.0

[MꢀH]^ꢀ; HPLC (method A): t_R=4.4 min (purity: 100%).

2’-(Methoxymethyl)-2-methylbiphenyl-4-carboxylic acid (56i):

Compound 56i was prepared following the procedure described

for 56d, starting from (2-methoxymethylphenyl)boronic acid. It

was isolated as a beige solid (1.74 g, 78%): ¹H NMR (300 MHz,

[D₆]DMSO): d=12.95 (brs, 1H), 7.92–7.91 (m, 1H), 7.84–7.81 (dd,

1H), 7.55–7.52 (m, 1H), 7.47–7.38 (m, 2H), 7.25–7.22 (m, 1H), 7.15–

7.14 (m, 1H), 4.09–4.06 (m, 2H), 3.17 (s, 3H), 2.07 ppm (s, 3H);

UHPLC/MS: m/z: 255.2 [MꢀH]^ꢀ; HPLC (method A): t_R=4.35 min

(purity: 96.8%).

2-(Methoxymethyl)-2’-methyl biphenyl-4-carboxylic acid (56a):

Step 1: To a solution of methyl 4-bromo-3-methylbenzoate (50 g,

218.27 mmol) in CHCl₃(1 L) under inert atmosphere were added N-

bromosuccinimide (NBS) (46.62 g, 261.93 mmol) in one portion and

AIBN (0.72 g, 4.37 mmol). The mixture was stirred at 708C for

2 days. The reaction mixture was cooled to RT and water (500 mL)

was added. The organic layer was separated and washed with satu-

rated aq NaHCO₃(50 mL), water (340 mL), brine (500 mL), dried

2-Ethoxy-2’-methyl-1,1’-biphenyl-4-carboxylic acid (56c): Step 1:

A mixture of methyl 4-bromo-3-hydroxybenzoate (CombiBlocks,

25 g, 108 mmol), o-tolylboronic acid (22 g, 162 mmol), anhydrous

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K₂CO₃(44 g, 324 mmol) and Pd(PPh₃)₄(6.25 g, 5.4 mmol) was dis-

solved in toluene (500 mL) and water (100 mL) under an inert at-

mosphere. The reaction mixture was heated at 1108C for 12 h. The

reaction mixture was cooled at RT, filtered through a Celite pad.

The filtrate was washed with 10% aq NaHCO₃(1ꢂ300 mL), water

(1ꢂ300 mL) and brine (2ꢂ300 mL). The organic layer was dried

over Na₂SO₄, filtered and concentrated in vacuo. The residue was

purified by chromatography (pet ether/EtOAc, 90:10!20:80), af-

fording methyl 2-hydroxy-2’-methyl-1,1’-biphenyl-4-carboxylate as

pale yellow solid (20 g, 77%): ¹H NMR (400 MHz, [D₆]DMSO): d=

9.85 (s, 1H), 7.52 (s, 1H), 7.45–7.43 (m, 1H), 7.25–7.20 (m, 3H),

7.15–7.13 (m, 1H), 7.11–7.09 (m, 1H), 3.84 (s, 3H), 2.09 ppm (s, 3H).

pared without further purification, in THF (60.00 mL), MeOH

(40.00 mL) and water (20.00 mL) was treated with LiOH (0.98 g,

23.29 mmol, 1.10 equiv). The reaction mixture was stirred at RT for

16 h. The solvents were removed under vacuo, affording com-

pound 73 as hygroscopic white foam that was used as a reagent

without further purification (19.9 mmol, 94%): ¹H NMR (300 MHz,

[D₆]DMSO): d=7.82 (s, 1H), 7.80–7.70 (m, 1H), 7.28–7.19 (m, 2H),

3.60 (s, 2H), 3.14 (s, 2H), 2.23 (s, 3H), 1.42 ppm (s, 9H); UHPLC/MS:

m/z: 280.3 [M+H]⁺. 278.2 [MꢀH]^ꢀ; HPLC (method A): t_R=2.05 min

(purity: 84.3%).

tert-Butyl N-{3-[(2-{[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-

carbonyl}hydrazino) carbonyl]benzyl}-N-methylglycinate (77):

Step 1: Oxalyl chloride (2.5 mL, 29.3 mmol, 1.5 equiv) and DMF

(30 mL, 0.4 mmol, 0.02 equiv) were added at RT to a solution of

56a (5.0 g, 19.5 mmol, 1.0 equiv) in dry toluene (100 mL). The reac-

tion mixture was stirred for 3 h and then concentrated in vacuo af-

fording the corresponding acyl chloride as a yellow oil (5.4 g, quan-

titative). The acyl chloride was immediately dissolved in CH₂Cl₂

(100 mL), and Et₃N (8.2 mL, 58.5 mmol, 3.0 equiv) was added fol-

lowed by hydrazinecarboxylic acid tert-butyl ester (2.8 g,

21.5 mmol, 1.1 equiv). The reaction was stirred at RT for 18 h.

Water (100 mL) was added, and the aqueous layer was separated

and extracted with CH₂Cl₂(2ꢂ100 mL). The combined organic ex-

tracts were washed with saturated aq NaHCO₃(75 mL), dried over

MgSO₄, filtered and concentrated to afford N’-(2-methoxymethyl-2’-

methyl-biphenyl-4-carbonyl)hydrazinecarboxylic acid tert-butyl

Step 2: To a stirred solution of methyl 2-hydroxy-2’-methyl-1,1’-bi-

phenyl-4-carboxylate (10 g, 41.2 mmol) in anhydrous CH₃CN

(100 mL) was added anhydrous K₂CO₃(17.1 g, 123.6 mmol) fol-

lowed by EtBr (15.4 mL, 206 mmol). The reaction mixture was

heated at 508C for 48 h, and was then cooled to RT and filtered.

The filtrate was concentrated in vacuo affording methyl 2-ethoxy-

2’-methyl-1,1’-biphenyl-4-carboxylate as a brown oil (11 g, 98%):

¹H NMR (400 MHz, [D₆]DMSO): d=7.70–7.68 (m, 1H), 7.62 (s, 1H),

7.29–7.16 (m, 5H), 4.09–4.03 (m, 2H), 3.95 (s, 3H), 2.15 (s, 3H),

1.35–1.25 ppm (m, 3H).

Step 3: To a stirred solution of methyl 2-ethoxy-2’-methyl-1,1’-bi-

phenyl-4-carboxylate (11 g, 40.6 mmol) in

a mixture of THF

(100 mL) and water (10 mL) was added LiOH (6.82 g, 162.7 mmol)

in portions. After 24 h at RT, the reaction mixture was evaporated,

and the residue was taken up in water (15 mL). This solution was

acidified to pH 1 with concentrated HCl and extracted with EtOAc

(2ꢂ20 mL). The organic layer was washed with brine (1ꢂ20 mL),

dried over Na₂SO₄, filtered and concentrated in vacuo affording

ester as

a

yellow oil (7.3 g, quantitative): ¹H NMR (300 MHz,

[D₆]DMSO): d=10.28 (s, 1H), 8.96 (s, 1H), 8.01 (s, 1H), 7.93–7.67 (m,

2H), 7.40–7.00 (m, 4H), 4.11 (s, 2H), 3.18 (s, 3H), 1.99 (s, 3H),

1.44 ppm (s, 9H); UHPLC/MS: m/z: 371.4 [M+H]⁺, 369.3 [MꢀH]^ꢀ;

HPLC (method A): t_R=4.34 min (purity: 98.0%).

56c as

a

pale yellow solid (9.7 g, 93%): ¹H NMR (400 MHz,

[D₆]DMSO): d=13.02 (brs, 1H), 7.57 (m, 2H), 7.25–7.18 (m, 4H),

7.11–7.08 (m, 1H), 4.06–4.01 (m, 2H), 2.05 (s, 3H), 1.18 ppm (m,

3H); UHPLC/MS: m/z: 255.0 [MꢀH]^ꢀ; HPLC (method B): t_R=

4.99 min (purity: 98.7%).

Step 2: A solution of N’-(2-methoxymethyl-2’-methyl-biphenyl-4-car-

bonyl)hydrazinecarboxylic acid tert-butyl ester (7.3 g, 19.5 mmol,

1.0 equiv) in CH₂Cl₂(100 mL) was cooled to 08C, and TFA (100 mL)

was then added dropwise. The reaction was stirred for 5 h at RT,

and then the solvents were removed in vacuo. The resulting yellow

oil was resuspended in CH₂Cl₂(50 mL) and water (50 mL). NaHCO₃

was added as solid until gas evolution ceased and the aqueous

layer reached pH 8. The organic layer was separated and washed

with water (1ꢂ50 mL) and brine (2ꢂ50 mL), dried over MgSO₄, fil-

tered and concentrated to afford compound 77 as a yellow oil

2-(Methoxymethyl)-2’-(trifluoromethyl)biphenyl-4-carboxylic

acid (56b): Compound 56b was prepared following the procedure

described for 56a, replacing o-tolylboronic acid with 2-(trifluoro-

methyl)phenylboronic acid in step 3. It was isolated as a pale

yellow solid (6.2 g, 74%): ¹H NMR (400 MHz, [D₆]DMSO): d=13.08

(s, 1H), 8.06 (s, 1H), 7.87–7.81 (m, 2H), 7.73–7.68 (m, 1H), 7.65–7.61

(m, 1H), 7.35 (d, J=7.5 Hz, 1H), 7.27 (d, J=7.9 Hz, 1H), 4.11–4.03

(m, 2H), 3.15 ppm (s, 3H); UHPLC/MS: m/z: 309.0 [MꢀH]^ꢀ; HPLC

(method A): t_R=4.65 min (purity: 99.6%).

1

(5.2 g, 98%): H NMR (300 MHz, CDCl₃): d=7.98–7.90 (m, 1H), 7.77

(dd, J=7.9, 1.9 Hz, 1H), 7.60 (s, 1H), 7.41–7.20 (m, 4H), 7.16–7.03

(m, 1H), 4.51–4.09 (m, 4H), 3.30 (s, 3H), 2.05 ppm (s, 3H); UHPLC/

MS: m/z: 271.3 [M+H]⁺, 269.3 [MꢀH]^ꢀ.

3-[(tert-Butoxycarbonylmethyl-methylamino)methyl]benzoic acid

lithium (73): Step 1: DIEA (11.14 mL, 65.48 mmol) was added to

a mixture of methyl 3-(bromomethyl)benzoate (5.00 g, 21.83 mmol)

and sarcosine tert-butyl ester hydrochloride (3965 mg, 21.83 mmol)

in CH₃CN (250 mL). The resulting solution was heated at 608C for

6 h. The solvents were evaporated, and water (75 mL) was added

to the residue. The mixture was extracted with EtOAc (2ꢂ75 mL).

The combined organic phases were washed with brine (40 mL),

dried over MgSO₄, filtered and concentrated in vacuo to afford

N’-Hydroxy-2-(methoxymethyl)-2’-methylbiphenyl-4-carboximi-

damide (75): Step 1: Oxalyl chloride (1.25 ml, 14.7 mmol) and DMF

(15 mL, 0.2 mmol) were added at RT to a solution of 56a (2.5 g,

9.75 mmol) in dry toluene (50 mL). The reaction mixture was stirred

for 3 h and then concentrated in vacuo. Toluene (50 mL) and 0.5m

NH₃in dioxane (106 mL, 53 mmol) were added, and the reaction

mixture was stirred at RT for 15 min. The solvents were removed in

vacuo, and the resulting white solid was washed with pentane

(200 mL), then water (200 mL) and pentane (2ꢂ100 mL) to afford

methyl

3-{[(2-tert-butoxy-2-oxoethyl)(methyl)amino]methyl}ben-

zoate as yellow oil (6.21 g, 97%): ¹H NMR (300 MHz, CDCl₃): d=

8.03–7.97 (m, 1H), 7.97–7.90 (m, 1H), 7.63–7.54 (m, 1H), 7.40 (t, J=

7.6 Hz, 1H), 3.91 (s, 3H), 3.72 (s, 2H), 3.18 (s, 2H), 2.36 (s, 3H),

1.56–1.37 ppm (m, 9H); UHPLC/MS: m/z: 294.4 [M+H]⁺; HPLC

(method A): t_R=2.53 min (purity: 93.1%).

2-(methoxymethyl)-2’-methylbiphenyl-4-carboxamide as

a white

1

solid (2.5 g, 91%): H NMR (300 MHz, [D₆]DMSO): d=8.05 (br s, 1H),

8.02 (d, J=1.5 Hz, 1H), 7.86–7.83 (m, 1H), 7.38 (br s, 1H), 7.32–7.22

(m, 3H), 7.18 (d, J=7.9 Hz, 1H), 7.08 (d, J=7.3 Hz, 1H), 4.10 (s, 2H),

3.16 (s, 3H), 1.99 ppm (s, 3H); UHPLC/MS: m/z: 256.2 [M+H]⁺,

254.2 [MꢀH]^ꢀ; HPLC (method A): t_R=3.47 min (purity: 98.5%).

Step 2: A solution of methyl 3-{[(2-tert-butoxy-2-oxoethyl)(methyl)-

amino]methyl}benzoate (6.2 g, 21.2 mmol, 1.00 equiv), used as pre-

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Step 2: To a mixture of 2-(methoxymethyl)-2’-methylbiphenyl-4-car-

boxamide (306 mg, 1.2 mmol, 1.0 equiv) in dioxane (6 mL) was

added pyridine (190 mL, 2.0 equiv). The solution was cooled to 08C

and trifluoroacetic anhydride (200 mL, 1.4 mmol, 1.2 equiv) was

added. The resulting solution was stirred at RT for 3 h. The reaction

mixture was diluted with Et₂O (50 mL) and washed with water

(50 mL). Aqueous layer was extracted with Et₂O (2ꢂ50 mL). The

combined organic layers were washed with saturated aq NaHCO₃

(2ꢂ100 mL) and brine (100 mL). The organic phase was then dried

over MgSO₄, filtered and concentrated to afford 2-(methoxymeth-

400 W). The reaction mixture was concentrated, EtOAc (50 mL) was

added to the residue, and the mixture was washed with 0.1n aq

HCl (2ꢂ25 mL), saturated aq NaHCO₃(25 mL), dried over MgSO₄,

and filtered. After evaporation of the solvents, the resulting crude

product was purified by flash chromatography (cyclohexane/

EtOAc, 9.5/0.5), affording methyl 4-[5-(2,2’-dimethylbiphenyl-4-yl)-

1,2,4-oxadiazol-3-yl]-2-fluorobenzoate as a white powder (495 mg,

1

34%): H NMR (300 MHz, [D₆]DMSO): d=8.17–8.16 (m, 1H), 8.12 (d,

J=7.2 Hz, 1H), 8.29–8.04 (m, 2H), 7.98 (dd, J=11.1, 2.5 Hz, 1H),

7.39–7.27 (m, 4H), 7.14–7.11 (m, 1H), 3.91 (s, 3H), 2.13 (s, 3H),

2.03 ppm (s, 3H); LC/MS: m/z: 403.0 [M+H]⁺; HPLC (method A):

t_R=6.23 min (purity: 93.5%).

yl)-2’-methylbiphenyl-4-carbonitrile

(190 mg,

67%): ¹H NMR

(300 MHz, [D₆]DMSO): d=7.89 (m, 1H), 7.84–7.80 (m, 1H), 7.35–

7.23 (m, 4H), 7.08 (d, J=7.3 Hz, 1H), 4.13 (d, J=12.8 Hz, 1H), 4.07

(d, J=12.8 Hz, 1H), 3.17 (s, 3H), 1.98 ppm (s, 3H); HPLC (meth-

od A): t_R=4.79 min (purity: 94.9%).

Step 2: Methyl 4-[5-(2,2’-dimethylbiphenyl-4-yl)-1,2,4-oxadiazol-3-

yl]-2-fluorobenzoate (495 mg, 1.23 mmol) was dissolved in THF

(10 mL) and MeOH (10 mL). 5m aq NaOH (1.23 mL, 6.15 mmol) was

added, and the mixture was stirred at RT overnight. 5n aq HCl

(1.23 mL, 6.15 mmol) was added, and the mixture was concentrat-

ed. The aqueous residue was diluted with CH₂Cl₂(50 mL) and was

washed with water (2ꢂ25 mL). The organic layer was dried over

MgSO₄, filtered and concentrated, affording compound 8 as a light

yellow solid (457 mg, 95%): ¹H NMR (300 MHz, [D₆]DMSO): d=

8.16–8.01 (m, 4H), 7.94 (dd, J=11.1, 1.4 Hz, 1H), 7.38–7.26 (m, 4H),

7.13–7.11 (m, 1H), 2.13 (s, 3H), 2.03 ppm (s, 3H); LC/MS: m/z: 386.9

[MꢀH]^ꢀ, 388.7 [M+H]⁺; HPLC (method A): t_R=5.38 min (purity:

98.0%); Anal. calcd for C₂₃H₁₇N₂O₃F: C 71.13, H 4.41, N 7.21, found:

C 71.26, H 4.75, N 6.83.

Step 3: Amidoxime 75 was prepared following the procedure de-

scribed for 52a, starting from 2-(methoxymethyl)-2’-methylbiphen-

1

yl-4-carbonitrile. It was isolated as white solid (1.9 g, 90%): H NMR

(300 MHz, [D₆]DMSO): d=9.64 (s, 1H), 7.82 (d, J=1.6 Hz, 1H), 7.65–

7.62 (m, 1H), 7.32–7.21 (m, 3H), 7.12–7.06 (m, 2H), 5.84 (br s, 2H),

4.08 (s, 2H), 3.15 (s, 3H), 2.00 ppm (s, 3H); UHPLC/MS: m/z: 271.2

[M+H]⁺; HPLC (method A): t_R=2.72 min (purity: 98.7%).

2-Fluoro-4-[5-(4-isobutyl-phenyl)-[1,2,4]oxadiazol-3-yl]-benzoic

acid (3): Step 1: 4-Isobutyl-benzoic acid (138.6 mg, 0.78 mmol), 1-

[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-

oxid hexafluorophosphate (HATU) (295.7 mg, 0.78 mmol) and DIEA

(0.27 mL, 1.56 mmol) were mixed in DMF (5 mL), and the reaction

mixture stirred at RT for 15 min. Compound 52a (150 mg,

0.71 mmol) was added, and the reaction mixture was stirred at RT

for 3 h. Toluene (5 mL) was added, and the reaction mixture was

stirred at 958C overnight. It was diluted with EtOAc (20 mL) and

washed with 1n aq HCl (20 mL). The resulting crude product was

purified by flash chromatography (EtOAc/cyclohexane, 10:90!

60:40), affording 2-fluoro-4-[5-(4-isobutyl-phenyl)-[1,2,4]oxadiazol-3-

yl]-benzoic acid methyl ester as colorless oil (100 mg, 40%):

¹H NMR (300 MHz, [D₆]DMSO): d=8.18–8.08 (m, 3H), 8.06–8.01 (m,

1H), 7.99–7.92 (m, 1H), 7.52–7.42 (d, J=7.9 Hz, 2H), 3.91 (s, 3H),

2.64–2.54 (d, J=7.1 Hz, 2H), 2.01–1.82 (m, 1H), 0.98–0.84 ppm (d,

J=6.7 Hz, 6H); UHPLC/MS: m/z: 355.4 [M+H]⁺; HPLC (method A):

t_R=6.14 min (purity: 92.3%).

4-[5-(4-Cyclohexyl-phenyl)-[1,2,4]oxadiazol-3-yl]-2-fluoro-benzoic

acid (4): Compound 4 was prepared following the procedure de-

scribed for compound 8, replacing carboxylic acid 56d with 4-cy-

clohexylbenzoic acid. It was isolated as a white solid (34.8 mg,

90%): ¹H NMR (300 MHz, [D₆]DMSO): d=13.55 (s, 1H), 8.17–7.97

(m, 3H), 7.95–7.87 (m, 2H), 7.6–7.45 (m, 2H), 2.6–2.8 (m, 1H), 1.92–

1.65 (m, 5H), 1.55–1.15 ppm (m, 5H); LC/MS: m/z: 367.0 [M+H]⁺;

HPLC (method A): t_R=5.55 min (purity: 99.6%).

4-(5-Biphenyl-4-yl-[1,2,4]oxadiazol-3-yl)-2-fluoro-benzoic acid (5):

Compound 5 was prepared following the procedure described for

compound 8, replacing carboxylic acid 56d with 4-phenylbenzoic

acid. It was isolated as an off-white solid (64 mg, 88%): ¹H NMR

(300 MHz, [D₆]DMSO): d=13.68 (s, 1H), 8.45–8.20 (m, 2H), 8.15–

7.95 (m, 5H), 7.9–7.75 (m, 2H), 7.70–7.35 ppm (m, 3H); LC/MS:

m/z: 361.0 [M+H]⁺; HPLC (method A): t_R=4.91 min (purity: 95.7%).

Step 2: 2-Fluoro-4-[5-(4-isobutyl-phenyl)-[1,2,4]oxadiazol-3-yl]-ben-

zoic acid methyl ester (100 mg, 0.28 mmol) was disolved in THF/

MeOH (5 mL, 1:1), and 5n aq NaOH (0.58 mL, 4.23 mmol) was

added. The reaction mixture stirred for 2 h at RT and was then con-

centrated. CH₂Cl₂(20 mL) was added, and the resulting solution

was washed with 5m aq HCl (20 mL), dried over MgSO₄, filtered

and concentrated, affording compound 3 as a white powder with

2-Fluoro-4-[5-(2-methylbiphenyl-4-yl)-1,2,4-oxadiazol-3-yl]benzo-

ic acid (6): Compound 6 was prepared following the procedure de-

scribed for compound 8, replacing carboxylic acid 56d with 3-

methyl-4-phenylbenzoic acid. It was isolated as an off-white solid

(64 mg, 88%): ¹H NMR (300 MHz, [D₆]DMSO): d=13.61 (s, 1H),

8.10–7.83 (m, 5H), 7.62–7.30 (m, 6H), 2.37 ppm (s, 3H); LC/MS:

m/z: 373.0 [MꢀH]^ꢀ; 375.0 [M+H]⁺; HPLC (method A): t_R=5.19 min

(purity: 99.3%).

1

acceptable purity (65 mg, 68%): H NMR (300 MHz, [D₆]DMSO): d=

8.17–8.05 (m, 3H), 8.04–7.99 (m, 1H), 7.96–7.88 (dd, J=11.1, 1.5 Hz,

1H), 7.53–7.39 (d, J=8.1 Hz, 2H), 2.63–2.54 (d, J=7.2 Hz, 2H),

2.02–1.83 (m, 1H), 0.96–0.81 ppm (d, J=6.6 Hz, 6H); UHPLC/MS:

m/z: 339.4 [MꢀH]^ꢀ, 341.4 [M+H]⁺; HPLC (method A): t_R=5.34 min

(purity: 99.8%).

2-Fluoro-4-[5-(2’-methylbiphenyl-4-yl)-1,2,4-oxadiazol-3-yl]ben-

zoic acid (7): Compound 7 was prepared following the procedure

described for compound 8, replacing carboxylic acid 56d with 4-

(2-methylphenyl)benzoic acid. It was isolated as an off-white solid

(36.8 mg, 98%): ¹H NMR (300 MHz, [D₆]DMSO): d=13.61 (s, 1H),

8.23–8.31 (m, 2H), 8.01–8.14 (m, 2H), 7.98–7.91 (m, 1H), 7.70–7.62

(m, 2H), 7.38–7.25 (m, 4H), 2.49 ppm (s, 3H); LC/MS: m/z: 373.0

[MꢀH]^ꢀ; 375.0 [M+H]⁺; HPLC (method A): t_R=5.17 min (purity:

97.6%).

4-[5-(2,2’-Dimethylbiphenyl-4-yl)-1,2,4-oxadiazol-3-yl]-2-fluoro-

benzoic acid (8): Step 1: Compound 56d (814 mg, 3.60 mmol), 52a

(637 mg, 3 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodii-

mide hydrochloride (690 mg, 3.60 mmol) were dissolved in THF

(10 mL) and CH₃CN (10 mL) in a 20 mL microwave vial under an

inert atmosphere. The reaction mixture was stirred overnight at RT.

DIEA (1.22 mL, 7.20 mmol) was added, and the mixture was heated

at 1508C for 30 min under microwave irradiation (full power,

2-Fluoro-4-[5-(2-methoxy-2’-methylbiphenyl-4-yl)-1,2,4-oxadia-

zol-3-yl]benzoic acid (9): Compound 9 was prepared following the

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procedure described for compound 8, replacing carboxylic acid

56d with 56e. It was isolated as an off-white solid (56 mg, 79%):

¹H NMR (300 MHz, [D₆]DMSO): d=13.62 (s, 1H), 8.15–8.02 (m, 2H),

7.97 (dd, J=11.1, 1.5 Hz, 1H), 7.88 (dd, J=7.7, 1.6 Hz, 1H), 7.82 (d,

J=1.6 Hz, 1H), 7.41 (d, J=7.8 Hz, 1H), 7.35–7.21 (m, 3H), 7.19–7.12

(m, 1H), 3.89 (s, 3H), 2.09 ppm (s, 3H); LC/MS: m/z: 403.0 [MꢀH]^ꢀ;

405.0 [M+H]⁺; HPLC (method A): t_R=5.14 min (purity: 96.5%).

300 mL), dried over MgSO₄, filtered and concentrated, affording

compound 13 as a beige solid (11.66 g, 75%): ¹H NMR (300 MHz,

[D₆]DMSO): d=13.64 (brs, 1H), 8.33 (d, J=1.41 Hz, 1H), 8.17 (dd,

J=7.92, 1.4 Hz, 1H), 8.13–8.03 (m, 2H), 7.96 (dd, J=11.10, 1.17 Hz,

1H), 7.43 (d, J=7.91 Hz, 1H), 7.37–7.27 (m, 3H), 7.15 (d, J=7.03 Hz,

1H), 4.25–4.14 (m, 2H), 3.25 (s, 3H), 2.04 ppm (s, 3H); UHPLC/MS:

m/z: 419.1 [M+H]⁺, 417.2 [MꢀH]^ꢀ; HPLC (method A): t_R=5.19 min

(purity: 99.2%).

2-Fluoro-4-{5-[2’-methyl-2-(trifluoromethyl)biphenyl-4-yl]-1,2,4-

oxadiazol-3-yl}benzoic acid (10): Compound 10 was prepared fol-

lowing the procedure described for compound 8, replacing carbox-

ylic acid 56d with 56 f. It was isolated as a white solid (109 mg,

2-Fluoro-4-{5-[2’-(methoxymethyl)-2-methylbiphenyl-4-yl]-1,2,4-

oxadiazol-3-yl}benzoic acid (14): Compound 14 was prepared fol-

lowing the procedure described for compound 8, replacing carbox-

ylic acid 56d with 56i. It was isolated as a white powder (88 mg,

84%): ¹H NMR (300 MHz, [D₆]DMSO): d=13.61 (brs, 1H), 8.17 (d,

J=1.0 Hz, 1H), 8.13–8.02 (m, 3H), 7.96 (dd, J=11.1, 1.3 Hz, 1H),

7.56–7.53 (m, 1H), 7.48–7.39 (m, 3H), 7.20–7.17 (m, 1H), 4.14 (d,

J=12.0 Hz, 1H), 4.09 (d, J=11.9 Hz, 1H), 3.13 (s, 3H), 2.14 ppm (s,

3H); UHPLC/MS: m/z: 417.2 [MꢀH]^ꢀ, 419.2 [M+H]⁺; HPLC (meth-

od A): t_R=5.38 min (purity: 98.2%).

1

85%): H NMR (300 MHz, [D₆]DMSO): d=13.62 (brs, 1H), 8.56–8.49

(m, 2H), 8.14–7.97 (m, 3H), 7.67 (d, J=8.0 Hz, 1H), 7.41–7.26 (m,

3H), 7.17 (d, J=7.4 Hz, 1H), 2.02 ppm (s, 3H); LC/MS: m/z: 440.9

[MꢀH]^ꢀ; HPLC (method A): t_R=5.51 min (purity: 99.0%).

2-Fluoro-4-[5-(2’-methoxy-2-methylbiphenyl-4-yl)-1,2,4-oxadia-

zol-3-yl]benzoic acid (11): Compound 11 was prepared following

the procedure described for compound 8, replacing carboxylic acid

56d with 56g. It was isolated as an off-white solid (45 mg, 98%):

¹H NMR (300 MHz, [D₆]DMSO): d=13.60 (brs, 1H), 8.14–7.99 (m,

4H), 7.98–7.90 (m, 1H), 7.47–7.36 (m, 2H), 7.21–7.11 (m, 2H), 7.10–

7.02 (m, 1H), 3.74 (s, 3H), 2.18 ppm (s, 3H); LC/MS: m/z: 403

[MꢀH]^ꢀ; 405.0 [M+H]⁺; HPLC (method A): t_R=5.11 min (purity:

97.1%).

N-(2-Fluoro-4-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzoyl)glycine (15): Step 1: To a solution of

compound 13 (2170 mg, 5.2 mmol) in dry CH₂Cl₂(40 mL), oxalyl

chloride (1.32 mL, 15.6 mmol) was added followed by dry DMF

(0.040 mL). The reaction mixture was stirred at RT for 2 h. It was

then concentrated to afford 2-fluoro-4-{5-[2-(methoxymethyl)-2’-

methylbiphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzoyl

chloride

as

2-Fluoro-4-{5-[2-methyl-2’-(trifluoromethyl)biphenyl-4-yl]-1,2,4-

oxadiazol-3-yl}benzoic acid (12): Compound 12 was prepared fol-

lowing the procedure described for compound 8, replacing carbox-

ylic acid 56d with 56h. It was isolated an off-white solid (75.9 mg,

89%): ¹H NMR (300 MHz, [D₆]DMSO): d=13.60 (brs, 1H), 7.97 (s,

1H), 7.95–7.80 (m, 3H), 7.79–7.66 (m, 2H), 7.63–7.53 (m, 1H), 7.53–

7.43 (m, 2H), 7.27–7.16 (m, 2H), 2.25 ppm (s, 3H); LC/MS: m/z:

441.0 [MꢀH]^ꢀ; 443.4 [M+H]⁺; HPLC (method A): t_R=5.40 min

(purity: 98.2%).

a white powder (2300 mg, 100%). Part of this product (125 mg,

0.29 mmol) was redissolved in dry THF (5 mL). DIEA (0.17 mL,

1 mmol) and glycine methyl ester hydrochloride (71.85 mg,

0.57 mmol) were added. The mixture was stirred overnight at RT.

The solvents were evaporated, and the crude residue was purified

by flash chromatography (cyclohexane/EtOAc, 90:10!40:60), af-

fording methyl N-(2-fluoro-4-{5-[2-(methoxymethyl)-2’-methylbi-

phenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzoyl)glycinate as an orange

solid (50 mg, 36%): ¹H NMR (300 MHz, CDCl₃): d=8.42 (m, 1H),

8.25 (t, J=8 Hz, 1H), 8.16 (dd, J=1.9, 7.9 Hz, 1H), 8.11 (dd, J=1.5,

7.9 Hz, 1H), 8.01 (dd, J=1.4, 12.4 Hz, 1H), 7.39–7.22 (m, 5H), 7.12

(m, 1H), 4.30 (d, J=4.8 Hz, 2H), 4.22 (d, J=2 Hz, 2H), 3.81 (s, 3H),

3.33 (s, 3H), 2.07 (s, 3H), 1.37 (d, J=6.8 Hz, 3H), 1.28 ppm (t, J=

7.1 Hz, 3H); UHPLC/MS: m/z: 490.2 [M+H]⁺488.3 [MꢀH]^ꢀ; HPLC

(method A): t_R=5.22 min (purity: 100%).

2-Fluoro-4-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-

oxadiazol-3-yl}benzoic acid (13): Step 1: Carboxylic acid 56a (4 g,

15.61 mmol) was dissolved in dry toluene (60 mL) under N₂at RT,

then oxalyl chloride (1.98 mL, 23.41 mmol) was added in one por-

tion followed by DMF (0.024 mL). The reaction mixture was stirred

at RT for 3 h. The reaction mixture was concentrated to afford

a yellow oil, which was then redissolved in toluene (40 mL) and

added dropwise at RT over 15 min to a mixture of amidoxime 52a

(3.31 g, 15.61 mmol) in pyridine (20 mL) and toluene (20 mL) under

an inert atmosphere. The reaction mixture was stirred at RT for

12 h, then heated at reflux for 24 h. The reaction mixture was

cooled to RT and concentrated, affording a beige solid. This solid

was suspended in MeOH (40 mL) and filtered, affording methyl 2-

fluoro-4-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-oxadia-

Step 2: To a solution of methyl N-(2-fluoro-4-{5-[2-(methoxymethyl)-

2’-methylbiphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzoyl)glycinate

(0.1806 mg, 0.369 mmol) in MeOH (4 mL), 5m aq NaOH (0.37 mL,

1.85 mmol) was added. The reaction was stirred at RT for 1 h.

EtOAc (20 mL) was added, and the organic phase was washed with

1n aq HCl (15 mL). The organic phase was dried over MgSO₄, fil-

tered, and concentrated to afford compound 15 as a white solid

(143.9 mg, 82%): ¹H NMR (300 MHz, [D₆]DMSO): d=(m, 1H), 8.34

(m, 1H), 8.19 (dd, J=1.9, 8 Hz, 1H), 8.07 (dd, J=1.5, 8 Hz, 1H), 7.98

(dd, J=1.5, 11 Hz, 1H), 7.91 (t, J=7.7 Hz, 1H), 7.44 (d, J=8 Hz, 1H),

7.39–7.24 (m, 3H), 7.15–7.11 (m, 1H), 4.23 (d, J=12.9 Hz, 1H), 4.17

(d, J=12.9 Hz, 1H), 3.96 (d, J=6 Hz, 2H), 3.26 (s, 3H), 2.04 ppm (s,

3H); UHPLC/MS: m/z: 476.1 [M+H]⁺474.3 [MꢀH]^ꢀ; HPLC (meth-

od A): t_R=4.63 min (purity: 99.9%).

1

zol-3-yl}benzoate as a beige solid (5.58 g, 82%): H NMR (300 MHz,

[D₆]DMSO): d=8.33 (m, 1H), 8.19–7.98 (m, 4H), 7.44–7.28 (m, 4H),

7.16–7.13 (m, 1H), 4.24–4.14 (m, 2H), 3.91 (s, 3H), 3.25 (s, 3H),

2.03 ppm (s, 3H); UHPLC/MS: m/z: 433.1 [M+H]⁺; HPLC (meth-

od A): t_R=5.94 min (purity: 94.9%).

Step 2: A solution of methyl 2-fluoro-4-{5-[2-(methoxymethyl)-2’-

methylbiphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzoate (16 g, 37 mmol)

in THF (400 mL) and MeOH (400 mL) at RT was treated with 5m aq

NaOH (37 mL, 184.99 mmol). The reaction mixture was stirred at RT

for 12 h. The reaction mixture was concentrated to give a yellow

solid. Water (600 mL) was added, and the aqueous phase was

washed with EtOAc (250 mL). The aqueous phase was then acidi-

fied to pH 2 with concentrated HCl and extracted with EtOAc (2ꢂ

300 mL). The combined organic phase was washed with brine (2ꢂ

N-(2-Fluoro-4-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzoyl)-b-alanine (16): Compound 16 was

prepared following the procedure described for compound 15, re-

placing glycine methyl ester hydrochloride with b-alanine methyl

ester hydrochloride. It was isolated as a white powder (1490.0 mg,

82%): ¹H NMR (300 MHz, [D₆]DMSO): d=8.58 (m, 1H), 8.33 (brs,

1H), 8.18 (dd, J=1.7, 8 Hz, 1H), 8.03 (dd, J=1.7, 8 Hz, 1H), 7.95 (d,

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J=10.6 Hz, 1H), 7.84 (t, J=7.6 Hz, 1H), 7.44 (d, J=7.9 Hz, 1H),

7.40–7.22 (m, 3H), 7.15 (m, 1H), 4.23 (d, J=12.9 Hz, 1H), 4.17 (d,

J=12.9 Hz, 1H), 3.49 (m, 2H), 3.25 (s, 3H), 2.53–2.45 (m, 2H),

2.04 ppm (s, 3H); UHPLC/MS: m/z: 490.3 [M+H]⁺488.4 [MꢀH]^ꢀ;

HPLC (method A): t_R=4.83 min (purity: 99.7%).

10.5 Hz, 1H), 7.59 (t, J=7.6 Hz, 1H), 7.30–7.34 (m, 3H), 7.25 (s, 1H),

7.13 (d, J=7.5 Hz, 1H), 4.80 (s, 2H), 4.23 (s, 2H), 4.22 (s, 2H), 3.33

(s, 3H), 2.08 ppm (s, 3H); UHPLC/MS: m/z: 463.3 [M+H]⁺; HPLC

(method A): t_R=5.77 min (purity: 97.9%).

[(3-{5-[2-(Methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-oxadia-

zol-3-yl}benzyl)oxy]acetic acid (21): Compound 21 was prepared

following the procedure described for compound 19, replacing

amidoxime 52b with 52d. It was isolated as a colorless oil

4-[(2-Fluoro-4-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzoyl)amino]butanoic acid (17): Com-

pound 17 was prepared following the procedure described for

compound 15, replacing glycine methyl ester hydrochloride with

methyl 4-aminobutyrate hydrochloride. It was isolated as a white

1

(298 mg, 82%): H NMR (300 MHz, [D₆]DMSO): d=12.75 (brs, 1H),

8.33 (d, J=1.5 Hz, 1H), 8.18 (dd, J=7.9, 1.8 Hz, 1H), 8.13 (brs, 1H),

8.06–8.02 (m, 1H), 7.61–7.55 (m, 2H), 7.43 (d, J=8.0H, 1H), 7.37–

7.26 (m, 3H), 7.15 (d, J=7.0 Hz, 1H), 4.68 (s, 2H), 4.23 (s, 2H), 4.17

(s, 2H), 3.25 (s, 3H), 2.04 ppm (s, 3H); LC/MS: m/z: 445.3 [M+H]⁺.

443.4 [MꢀH]^ꢀ; HPLC (method A): t_R=5.18 min (purity: 97.5%).

1

solid (135 mg, 52%): H NMR (300 MHz, [D₆]DMSO): d=12.10 (brs,

1H), 8.57–8.53 (m, 1H), 8.33 (brs, 1H), 8.19 (dd, J=1.8, 7.9 Hz, 1H),

8.03 (dd, J=1.5, 7.9 Hz, 1H), 7.95 (dd, J=1.3, 10.5 Hz, 1H), 7.82 (t,

J=7.5 Hz, 1H), 7.43 (d, J=7.9 Hz, 1H), 7.39–7.24 (m, 3H), 7.15–7.11

(m, 1H), 4.23 (d, J=12.9 Hz, 1H), 4.17 (d, J=12.9 Hz, 1 J), 3.30–3.22

(m, 2H), 3.25 (s, 3H), 3.21–3.13 (m, 2H), 2.31 (t, J=7.3 Hz, 2H), 2.04

(s, 3H), 1.77 ppm (q, J=7.3 Hz, 2H); UHPLC/MS: m/z: 504.2

[M+H]⁺502.3 [MꢀH]^ꢀ; HPLC (method A): t_R=4.8 min (purity:

99%).

N-(4-{5-[2-(Methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-oxadia-

zol-3-yl}benzyl)glycine, hydrochloride salt (22): Compound 22

was prepared following the procedure described for compound 3

(step 1), replacing carboxylic acid 56d with 56a and amidoxime

52a with 52e. The resulting N-Boc-protected tert-butyl ester deriv-

ative was treated with 4m HCl solution in dioxane similarly to the

procedure described for compound 19 (step 2), affording com-

pound 22 as a white powder (172 mg, 84%): ¹H NMR (300 MHz,

[D₆]DMSO): d=8.33 (d, J=1.5 Hz, 1H), 8.21–8.15 (m, 3H), 7.77 (d,

J=8.5 Hz, 2H), 7.44 (d, J=8.1 Hz, 1H), 7.39–7.27 (m, 3H), 7.15 (d,

J=7.0 Hz, 1H), 4.28 (s, 2H), 4.26–4.14 (m, 2H), 3.90 (s, 2H), 3.26 (s,

3H), 2.04 ppm (s, 3H); UHPLC/MS: m/z: 442.1 [MꢀH]^ꢀ, 444.0

[M+H]⁺; HPLC (method A): t_R=3.96 min (purity: 99.7%).

3-({2-Fluoro-4-[5-(2-methoxymethyl-2’-methyl-biphenyl-4-yl)-

[1,2,4]oxadiazol-3-yl]-benzoyl}-methylamino)propionic acid (18):

Compound 18 was prepared following the procedure described

for compound 15, replacing glycine methyl ester hydrochloride

with methyl N-methyl-b-alaninate hydrochloride. It was isolated as

a white solid (42 mg, 97%): ¹H NMR (300 MHz, [D₆]DMSO): d=

12.37 (s, 1H), 8.34 (d, J=2.3 Hz, 1H), 8.18 (dd, J=8.0, 2.0 Hz, 1H),

8.10–7.92 (m, 2H), 7.69–7.59 (m, 1H), 7.44 (d, J=8.0 Hz, 1H), 7.40–

7.23 (m, 3H), 7.15 (d, J=7.3 Hz, 1H), 4.32–4.07 (m, 2H), 3.70 (t, J=

7.2 Hz, 1H), 3.43 (t, J=7.2 Hz, 1H), 3.25 (s, 3H), 3.02 (s, 1H), 2.90 (s,

2H), 2.61 (t, J=7.2 Hz, 1H), 2.54–2.50 (m, 1H), 2.04 ppm (s, 3H);

UHPLC/MS: m/z: 504.2 [M+H]⁺. 502.2 [MꢀH]^ꢀ; HPLC (method A):

t_R=4.84 min (purity: 98.3%).

N-(4-{5-[2-(Methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-oxadia-

zol-3-yl}benzyl)-N-methylglycine, hydrochloride salt (23): Com-

pound 23 was prepared following the procedure described for

compound 22, replacing amidoxime 52e with 52 f. It was isolated

as a white powder (168 mg, 86%): ¹H NMR (300 MHz, [D₆]DMSO):

d=8.33 (d, J=1.4 Hz, 1H), 8.24–8.15 (m, 3H), 7.79 (s, 1H), 7.77 (s,

1H), 7.44 (d, J=7.9 Hz, 1H), 7.38–7.27 (m, 3H), 7.15 (d, J=7.0 Hz,

1H), 4.43 (s, 2H), 4.26–4.14 (m, 2H), 4.06 (s, 2H), 3.26 (s, 3H), 2.78

(s, 3H), 2.04 ppm (s, 3H); UHPLC/MS: m/z: 456.1 [MꢀH]^ꢀ, 458.0

[M+H]⁺; HPLC (method A): t_R=4.01 min (purity: 99.5%).

(2-Fluoro-4-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-

oxadiazol-3-yl}phenoxy)acetic acid (19): Step 1: Using the proce-

dure described for compound 8 (step 1), replacing the carboxylic

acid 56d with 56a and amidoxime 52a with 52b, tert-butyl (2-

fluoro-4-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-oxadia-

zol-3-yl}phenoxy)acetate was isolated as a white powder (167 mg,

66%): ¹H NMR (400 MHz, CDCl₃): d=8.41 (d, J=1.3 Hz, 1H), 8.15

(dd, J=7.8, 1.8 Hz, 1H), 7.98–7.91 (m, 2H), 7.36–7.23 (m, 4H), 7.13

(d, J=7.1 Hz, 1H), 7.03–6.97 (m, 1H), 4.68 (s, 2H), 4.22 (s, 2H), 3.33

(s, 3H), 2.07 (s, 3H), 1.49 ppm (s, 9H); UHPLC/MS: m/z: 505.2

[M+H]⁺; HPLC (method A): t_R=6.36 min (purity: 98.6%).

N-(4-{5-[2-(Methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-oxadia-

zol-3-yl}benzyl)-N-methyl-b-alanine, hydrochloride salt (24):

Compound 24 was prepared following the procedure described

for compound 22, replacing amidoxime 52e with 52g. It was iso-

lated as

a

white powder (37 mg, 54%): ¹H NMR (300 MHz,

[D₆]DMSO): d=8.33 (s, 1H), 8.25–8.15 (m, 3H), 7.84 (d, J=8.0 Hz,

2H), 7.47–7.27 (m, 4H), 7.15 (d, J=7.1 Hz, 1H), 4.45 (s, 2H), 4.26–

4.14 (m, 2H), 3.41–3.23 (m, 5H), 2.88 (t, J=7.2 Hz, 2H), 2.69 (s, 3H),

2.04 ppm (s, 3H); UHPLC/MS: m/z: 470.2 [MꢀH]^ꢀ, 472.1 [M+H]⁺;

HPLC (method A): t_R=4.03 min (purity: 99.1%).

Step 2: To tert-butyl (2-fluoro-4-{5-[2-(methoxymethyl)-2’-methylbi-

phenyl-4-yl]-1,2,4-oxadiazol-3-yl}phenoxy)acetate

(116 mg,

0.23 mmol), obtained in step 1, was added a 4m HCl in dioxane

(2 mL, 8 mmol). The mixture was stirred at RT for 16 h. The solvents

were concentrated to dryness to afford compound 19 as a white

powder (98 mg, 95%): ¹H NMR (300 MHz, [D₆]DMSO): d=13.24

(brs, 1H), 8.31 (s, 1H), 8.16 (d, J=8.2 Hz, 1H), 7.92 (s, 1H), 7.89 (s,

1H), 7.42 (d, J=8.0 Hz, 1H), 7.35–7.30 (m, 4H), 7.15 (d, J=7.1 Hz,

1H), 4.93 (s, 2H), 4.25–4.14 (m, 2H), 3.25 (s, 3H), 2.04 ppm (s, 3H);

UHPLC/MS: m/z: 447.2 [MꢀH]^ꢀ, 449.1 [M+H]⁺; HPLC (method A):

t_R=5.18 min (purity: 100.0%); Anal. calcd for C₂₅H₂₁N₂O₅F·0.1H₂O: C

66.69, H 4.75, N 6.22, found: C 66.53, H 4.71, N 6.18.

N-(3-{5-[2-(Methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-oxadia-

zol-3-yl}benzyl)glycine, hydrochloride salt (25): Compound 25

was prepared following the procedure described for compound

22, replacing amidoxime 52e with 52h. It was isolated as a white

1

powder (250 mg, 78%): H NMR (300 MHz, [D₆]DMSO): d=8.34 (dd,

J=6.3, 1.8 Hz, 2H), 8.18 (dq, J=7.8, 1.8 Hz, 2H), 7.81–7.63 (m, 2H),

7.45 (d, J=8.0 Hz, 1H), 7.37 (dd, J=5.1, 1.4 Hz, 2H), 7.35–7.25 (m,

1H), 7.20–7.09 (m, 1H), 4.31 (s, 2H), 4.28–4.11 (m, 2H), 3.89 (s, 2H),

3.26 (s, 3H), 2.04 ppm (s, 3H); UHPLC/MS: m/z: 442.3 [MꢀH]^ꢀ,

444.3 [M+H]⁺; HPLC (method A): t_R=3.85 min (purity: 96.8%).

{2-Fluoro-4-[5-(2-methoxymethyl-2’-methyl-biphenyl-4-yl)-

[1,2,4]oxadiazol-3-yl]-benzyloxy}-acetic acid (20): Compound 20

was prepared following the procedure described for compound

19, replacing amidoxime 52b with 52c. It was isolated as a dark

yellow oil (61.3 mg, 20%): ¹H NMR (400 MHz, CDCl₃): d=8.42 (s,

1H), 8.16 (d, J=7.4 Hz, 1H), 8.01 (d, J=7.9 Hz, 1H), 7.92 (d, J=

N-(3-{5-[2-(Methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-oxadia-

zol-3-yl}benzyl)-N-methylglycine, hydrochloride salt (26): Com-

pound 26 was prepared following the procedure described for

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compound 13 (step 1), replacing amidoxime 52a with 52i. The re-

sulting tert-butyl ester was hydrolysed with 4m HCl in dioxane sim-

ilarly to the procedure described for compound 19 (step 2), afford-

ing compound 26 as a white powder (1.4 g, 85%): ¹H NMR

(300 MHz, [D₆]DMSO): d=8.35–8.34 (m, 2H), 8.22 (d, J=1.8 Hz,

1H), 8.17 (dd, J=7.9, 1.8 Hz, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.72 (t,

J=7.7 Hz, 1H), 7.44 (d, J=8.0 Hz, 1H), 7.37–7.27 (m, 3H), 7.15 (d,

J=7.2 Hz, 1H), 4.52 (s, 2H), 4.25–4.13 (m, 4H), 3.26 (s, 3H), 2.82 (s,

3H), 2.04 ppm (s, 3H); UHPLC/MS: m/z: 456.4 [MꢀH]^ꢀ, 458.4

[M+H]⁺; HPLC (method A): t_R=4.45 min (purity: 98.6%); Anal.

calcd for C₂₇H₂₈O₄N₃Cl: C 65.65, H 5.71, N 8.51, Cl 7.18, found: C

65.36, H 5.65, N 8.49, Cl 7.08.

(29): Compound 29 was prepared following the procedure de-

scribed for compound 28, replacing amidoxime 52k with 75 and

carboxylic acid 56a with 73. It was isolated as a white powder

1

(163 mg, 88%): H NMR (300 MHz, [D₆]DMSO): d=8.47 (t, J=1.7 Hz,

1H), 8.32 (dt, J=7.9, 1.3 Hz, 1H), 8.28–8.22 (m, 1H), 8.08 (dd, J=

7.9, 1.8 Hz, 1H), 7.92 (dt, J=7.8, 1.4 Hz, 1H), 7.78 (t, J=7.8 Hz, 1H),

7.45–7.23 (m, 4H), 7.21–7.06 (m, 1H), 4.54 (brs, 2H), 4.32–4.02 (m,

4H), 3.24 (s, 3H), 2.83 (s, 3H), 2.04 ppm (s, 3H); UHPLC/MS: m/z:

458.4 [M+H]⁺, 456.5 [MꢀH]^ꢀ; HPLC (method A): t_R=4.01 min

(purity: 99.4%).

tert-Butyl N-{3-[(2-{[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-

carbonyl}hydrazino)carbonyl]benzyl}-N-methylglycinate (78): To

a solution of 73 (2.9 g, 10.0 mmol) in DMF (30 mL) was added DIEA

(5.2 mL, 30.0 mmol) and HATU (5.7 g, 15.0 mmol). After 10 min at

RT, intermediate 77 (2.7 g, 10.0 mmol) was added and the mixture

was stirred at RT for 3 h. Water was added (30 mL), and the mixture

was extracted with EtOAc (3ꢂ20 mL). The combined organic layers

were washed with brine (3ꢂ20 mL), dried over MgSO₄, filtered and

concentrated in vacuo. The orange oily residue was purified by

flash chromatography (heptane/EtOAc, 60:40) affording 78 as

a yellow oil (1.8 g, 35%): ¹H NMR (300 MHz, [D₆]DMSO): d=10.57

(d, J=13.1 Hz, 2H), 8.19–8.02 (m, 1H), 7.98–7.77 (m, 3H), 7.60–7.42

(m, 2H), 7.40–7.19 (m, 4H), 7.12 (dd, J=8.1, 1.3 Hz, 1H), 4.14 (s,

2H), 3.72 (s, 2H), 3.28–3.13 (m, 5H), 2.28 (s, 3H), 2.02 (s, 3H),

1.44 ppm (s, 9H); UHPLC/MS: m/z: 530.6 [MꢀH]^ꢀ, 532.6 [M+H]⁺;

HPLC (method A): t_R=3.62 min (purity: 89.8%).

N-(3-{5-[2-(Methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-oxadia-

zol-3-yl}benzyl)-N-methyl-b-alanine, hydrochloride salt (27):

Compound 27 was prepared following the procedure described

for compound 26, replacing amidoxime 52i with 52j. It was isolat-

ed as an off-white powder (60 mg, 26%): ¹H NMR (300 MHz,

[D₆]DMSO): d=12.74 (br s, 1H), 10.38 (br s, 1H), 8.36–8.33 (m, 1H),

8.23–8.20 (m, 1H), 8.18–8.15 (m, 1H), 7.87–7.84 (m, 1H), 7.72 (t, J=

7.8 Hz, 1H), 7.44 (d, J=7.9 Hz, 1H), 7.37–7.28 (m, 3H), 7.15–7.13

(m, 1H), 4.48 (br s, 2H), 4.23 (d, J=12.7 Hz, 1H), 4.16 (d, J=

12.7 Hz, 1H), 3.25 (s, 3H), 2.89–2.84 (m, 2H), 2.70 (s, 3H), 2.03 ppm

(s, 3H); UHPLC/MS: m/z: 472.4 [M+H]⁺; HPLC (method A): t_R=

4.53 min (purity: 97.4%).

N-(2-{5-[2-(Methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-oxadia-

zol-3-yl}benzyl)-N-methylglycine, hydrochloride salt (28): Step 1:

HATU (217 mg, 0.57 mmol) was added to a solution of carboxylic

acid 56a (154 mg, 0.60 mmol) and DIEA (0.215 mL, 1.26 mmol) in

anhydrous DMF (5 mL) at 08C. The resulting mixture was stirred at

08C for 15 min. Amidoxime 52k (176 mg, 0.60 mmol) was added in

one portion, and the mixture was stirred 2 h at 08C. The reaction

was diluted with EtOAc (10 mL) and washed with water (2ꢂ5 mL)

and brine (3ꢂ5 mL). The organic layer was dried over MgSO₄, fil-

tered and concentrated in vacuo. The residue was taken up in

CH₃CN (5 mL) and heated at 1508C for 30 min under microwave ir-

radiation (full power, 400 W). The reaction mixture was concentrat-

ed in vacuo. After purification by flash chromatography (cyclohex-

ane/EtOAc, 90:10!40:60), tert-butyl N-(2-{5-[2-(methoxymethyl)-2’-

methylbiphenyl-4-yl]-1,2,4-oxadiazol-3-yl}benzyl)-N-methylglycinate

was obtained as a brown oil (127 mg, 41%): ¹H NMR (300 MHz,

[D₆]DMSO): d=8.31 (d, J=1.5 Hz, 1H), 8.16 (dd, J=7.9, 1.9 Hz, 1H),

7.91 (dd, J=7.6, 1.3 Hz, 1H), 7.67–7.56 (m, 2H), 7.49 (dt, J=7.4,

1.6 Hz, 1H), 7.42 (d, J=7.9 Hz, 1H), 7.38–7.26 (m, 3H), 7.15 (d, J=

7.0 Hz, 1H), 4.25–4.13 (m, 2H), 4.06 (s, 2H), 3.24 (s, 3H), 3.17 (s,

2H), 2.22 (s, 3H), 2.04 (s, 3H), 1.41 ppm (s, 9H); UHPLC/MS: m/z:

514.1 [M+H]⁺; HPLC (method A): t_R=4.83 min (purity: 90.3%).

({3-[5-(2-Methoxymethyl-2’-methyl-biphenyl-4-yl)-[1,3,4]oxadia-

zol-2-yl]-benzyl}-methylamino)acetic acid, hydrochloride salt

(30): Step 1: Compound 78 (200 mg, 0.38 mmol, 1.0 equiv) was dis-

solved in CH₂Cl₂(5.00 mL) and 1-methylimidazole (90 mL, 1.1 mmol)

was added. The reaction mixture was cooled to 08C, and then tri-

fluoromethanesulfonic anhydride (93 mL, 0.56 mmol) was added.

The solution was stirred at RT for 2 h. Water (5 mL) was added, and

the aqueous layer was extracted with CH₂Cl₂(2ꢂ5 mL). The com-

bined organic extracts were dried over Na₂SO₄, filtered and con-

centrated. The crude product was purified by flash chromatogra-

phy (heptane/EtOAc, 90:10!40:60) affording ({3-[5-(2-methoxy-

methyl-2’-methyl-biphenyl-4-yl)-[1,3,4]oxadiazol-2-yl]-benzyl}methy-

lamino)acetic acid tert-butyl ester as a yellow oil (134 mg, 69%):

¹H NMR (300 MHz, CDCl₃): d=8.37–8.30 (m, 1H), 8.20–8.03 (m, 3H),

7.67–7.58 (m, 1H), 7.58–7.47 (m, 1H), 7.38–7.21 (m, 4H), 7.14 (d,

J=7.1 Hz, 1H), 4.23 (s, 2H), 3.87 (s, 2H), 3.38–3.24 (m, 5H), 2.48 (s,

3H), 2.08 (s, 3H), 1.50 ppm (s, 9H); UHPLC/MS: m/z: 514.6 [M+H]⁺;

HPLC (method A): t_R=4.32 min (purity: 97.6%).

Step 2: Hydrolysis of ({3-[5-(2-methoxymethyl-2’-methyl-biphenyl-4-

yl)-[1,3,4]oxadiazol-2-yl]-benzyl}methylamino)acetic acid tert-butyl

ester was performed following the procedure described for com-

pound 28 (step 2), affording compound 30 as a white powder

Step 2: A solution of tert-butyl N-(2–5-[2-(methoxymethyl)-2’-meth-

ylbiphenyl-4-yl]-1,2,4-oxadiazol-3-ylbenzyl)-N-methylglycinate

(103 mg, 0.20 mmol) in 4n HCl in dioxane (2.5 mL, 10 mmol) was

stirred at RT for 7 h. The reaction mixture was concentrated in va-

cuo. The residue was taken up in CH₃CN (2 mL), and a white solid

precipitated out. The precipitate was isolated by filtration and

dried in vacuo to give compound 28 as a white powder (69 mg,

70%): ¹H NMR (300 MHz, [D₆]DMSO): d=8.36 (d, J=1.4 Hz, 1H),

8.22 (dd, J=7.9, 1.7 Hz, 1H), 8.18–8.13 (m, 1H), 7.91–8.87 (m, 1H),

7.78–7.71 (m, 2H), 7.45 (d, J=7.8 Hz, 1H), 7.39–7.27 (m, 3H), 7.15

(d, J=7.1 Hz, 1H), 4.87 (s, 2H), 4.26–4.14 (m, 4H), 3.25 (s, 3H), 2.89

(s, 3H), 2.04 ppm (s, 3H); UHPLC/MS: m/z: 456.1 [MꢀH]^ꢀ, 458.0

[M+H]⁺; HPLC (method A): t_R=3.90 min (purity: 99.4%).

1

(88 mg, 68%): H NMR (300 MHz, [D₆]DMSO): d=8.36–8.24 (m, 2H),

8.24–8.17 (m, 1H), 8.12 (dd, J=7.9, 1.9 Hz, 1H), 7.82–7.63 (m, 2H),

7.46–7.23 (m, 4H), 7.15 (d, J=7.2 Hz, 1H), 4.30 (s, 2H), 4.26–4.09

(m, 2H), 3.88 (s, 2H), 3.24 (s, 3H), 2.68 (s, 3H), 2.04 ppm (s, 3H);

UHPLC/MS: m/z: 458.5 [M+H]⁺, 456.6 [MꢀH]^ꢀ; HPLC (method A):

t_R=3.63 min (purity: 99.5%).

{3-[5-(2-Methoxymethyl-2’-methyl-biphenyl-4-yl)-[1,3,4]thiadia-

zol-2-yl]-benzyl}methylamino)acetic acid, hydrochloride salt (31):

Step 1: Compound 78 (200 mg, 0.38 mmol) was dissolved in dry

THF (8.00 mL) and Lawesson’s reagent (160 mg, 0.39 mmol) was

added in one portion. The resulting suspension was then heated at

1208C for 30 min under microwave irradiation (full power, 400 W).

The reaction mixture was passed through a column of neutral alu-

({3-[3-(2-Methoxymethyl-2’-methyl-biphenyl-4-yl)-[1,2,4]oxadia-

zol-5-yl]-benzyl}-methylamino)acetic acid, hydrochloride salt

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mina (15 g), eluting with EtOAc. The solvents were evaporated in

vacuo. The crude product was purified by flash chromatography

(heptane/EtOAc, 90:10!40:60) affording ({3-[5-(2-methoxymethyl-

2’-methyl-biphenyl-4-yl)-[1,3,4]thiadiazol-2-yl]-benzyl}methylamino)-

methyl)-2’-methylbiphenyl-4-yl)-1,2,4-oxadiazol-3-yl)phenyl)metha-

nol was isolated as a white solid (80 mg, 65%): H NMR (400 MHz,

1

CDCl₃): d=8.43 (d, J=1.8 Hz, 1H), 8.24–8.14 (m, 2H), 7.36–7.21 (m,

6H), 7.13 (d, J=7.5 Hz, 1H), 4.81 (d, J=5.7 Hz, 2H), 4.23 (t, J=

13.4 Hz, 2H), 3.32 (s, 3H), 2.08 (s, 3H), 1.90 ppm (t, J=6.0 Hz, 1H);

LC/MS: m/z: 405 [M+H]⁺; HPLC (method C): t_R=4.25 min (purity:

99.1%).

1

acetic acid tert-butyl ester as a yellow oil (100 mg, 52%): H NMR

(300 MHz, CDCl₃): d=8.16 (dd, J=1.9, 0.7 Hz, 1H), 8.09–7.91 (m,

3H), 7.61–7.51 (m, 1H), 7.51–7.41 (m, 1H), 7.36–7.21 (m, 4H), 7.19–

7.09 (m, 1H), 4.23–4.19 (m, 2H), 3.78 (s, 2H), 3.32 (s, 3H), 3.23 (s,

2H), 2.41 (s, 3H), 2.10 (s, 3H), 1.54–1.44 ppm (m, 9H); UHPLC/MS:

m/z: 530.6 [M+H]⁺; HPLC (method A): t_R=4.48 min (purity:

100.0%).

Step 2: (3-fluoro-4-(5-(2-(methoxymethyl)-2’-methylbiphenyl-4-yl)-

1,2,4-oxadiazol-3-yl)phenyl)methanol (80 mg, 0.2 mmol) was dis-

solved in dioxane (5 mL) and MnO₂(200 mg, 2.32 mmol) was

added. The mixture was heated at 708C overnight, and then the

solvent was removed in vacuo. The residue was triturated with

a mixture of petroleum ether and Et₂O to give 4-(5-(2-(methoxy-

methyl)-2’-methylbiphenyl-4-yl)-1,2,4-oxadiazol-3-yl)-3-fluorobenzal-

dehyde as an off-white solid (55.9 mg, 69%). This intermediate was

dissolved in a mixture of MeOH (3 mL), CH₂Cl₂(3 mL) and acetic

acid (75 mL), and the solution was treated with NaBH₃CN (9.5 mg,

0.15 mmol) and 2-(methylamino)acetic acid (21 mg, 0.28 mmol).

The mixture was stirred at RT overnight, and then the solvent was

then removed in vacuo. The residue was purified by preparative

HPLC (see general comments for details), treated with 4m HCl in

dioxane (3 mL) and triturated with CH₃CN, affording compound 34

Step 2: Hydrolysis of ({3-[5-(2-methoxymethyl-2’-methyl-biphenyl-4-

yl)-[1,3,4]thiadiazol-2-yl]-benzyl}methylamino)acetic acid tert-butyl

ester was performed following the procedure described for com-

pound 28 (step 2), affording compound 31 as a white powder

1

(76 mg, 79%): H NMR (300 MHz, [D₆]DMSO): d=8.32 (t, J=1.7 Hz,

1H), 8.23–8.09 (m, 2H), 8.01 (dd, J=7.9, 2.0 Hz, 1H), 7.81–7.64 (m,

2H), 7.41–7.23 (m, 4H), 7.18–7.08 (m, 1H), 4.49 (s, 2H), 4.31–4.04

(m, 4H), 3.24 (s, 3H), 2.82 (s, 3H), 2.05 ppm (s, 3H); UHPLC/MS: m/

z: 474.5 [M+H]⁺, 472.5 [MꢀH]^ꢀ; HPLC (method A): t_R=3.80 min

(purity: 99.8%).

N-(3-{3-[2-(Methoxymethyl)-2’-methylbiphenyl-4-yl]-1H-1,2,4-tria-

zol-5-yl}benzyl)-N-methylglycine, hydrochloride salt (32): Step 1:

Using the procedure described for the preparation of 52 f (step 1),

starting from 3-(bromomethyl)benzonitrile (5.9 g, 30.3 mmol) and

sarcosine tert-butyl ester hydrochloride (5.0 g, 27.5 mmol), tert-

butyl N-(3-cyanobenzyl)-N-methylglycinate was isolated as a brown

oil (7 g, 98%): HPLC (method A): t_R=2.60 min (purity: 90.5%).

1

as a white solid (55.4 mg, 78%): H NMR (400 MHz, [D₆]DMSO): d=

8.31 (d, J=1.8 Hz, 1H), 8.24 (t, J=7.7 Hz, 1H), 8.18 (dd, J=7.9,

1.9 Hz, 1H), 7.66 (d, J=11.3 Hz, 1H), 7.60 (dd, J=8.1, 1.6 Hz, 1H),

7.43 (d, J=7.9 Hz, 1H), 7.37–7.26 (m, 3H), 7.13 (d, J=7.4 Hz, 1H),

4.42 (s, 2H), 4.24–4.12 (m, 2H), 4.08 (s, 2H), 3.23 (s, 3H), 2.81 ppm

(s, 3H), 2.02 (s, 3H); LC/MS: m/z: 476 [M+H]⁺; HPLC (method C):

t_R=3.08 min (purity: 99.4%).

Step 2: In a microwave vial, K₂CO₃(767 mg, 5.6 mmol) was added

to a solution of intermediate 77 (300 mg, 1.1 mmol) and tert-butyl

N-(3-cyanobenzyl)-N-methylglycinate (578 mg, 2.2 mmol) in nBuOH

(6 mL). The resulting mixture was heated at 2008C for 2 h under

microwave irradiation (full power, 400 W). Water (5 mL) was added,

and the solution was directly purified by preparative HPLC (see

general comments for details). Pure fractions were isolated, mixed

and concentrated. The residue was dissolved in CH₃CN (5 mL) and

2.5m HCl in Et₂O (5 mL) was added. The mixture was stirred over-

night at RT. The solvents were removed, and the solid was sus-

pended in Et₂O (2 mL) and filtered affording compound 32 as an

orange powder (80 mg, 15%): ¹H NMR (300 MHz, [D₆]DMSO): d=

8.22 (s, 1H), 8.17–8.13 (m, 2H), 8.00 (dd, J=1.7, 8.0 Hz, 1H), 7.66–

7.58 (m, 2H), 7.31–7.21 (m, 4H), 7.07 (d, J=6.8 Hz, 1H), 4.41 (br s,

2H), 4.13 (s, 2H), 4.07 (s, 2H), 3.16 (s, 3H), 2.81 (s, 3H), 1.98 ppm (s,

3H); UHPLC/MS: m/z: 457.2 [M+H]⁺, 455.3 [MꢀH]^ꢀ; HPLC (meth-

od A): t_R=3.81 min (purity: 96.2%).

2-((4-(5-(2-(Methoxymethyl)-2’-methylbiphenyl-4-yl)-1,2,4-oxa-

diazol-3-yl)-2-(trifluoromethyl)benzyl)(methyl)amino)acetic acid

(35): To a solution of carboxylic acid 56a (0.112 g, 0.31 mmol) and

amidoxime 52n (0.067 mg, 0.26 mmol) in CH₃CN (2 mL) was added

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride

(0.069 g, 0.36 mmol). The reaction mixture was stirred at RT for

18 h. Pyridine (2 mL) was added to the mixture, and the reaction

was heated at 1508C for 30 min under microwave irradiation (full

power, 400 W). The reaction was repeated at the same scale and

combined for work up. The solvent was removed in vacuo, and the

residue was redissolved in CH₂Cl₂(15 mL). The mixture was washed

with water (10 mL) and brine (10 mL), dried over MgSO₄and fil-

tered. The solvent was evaporated in vacuo, and the residue was

purified by flash chromatography (heptane/EtOAc, 90:10!40:60).

The crude material was treated with a 4n HCl in dioxane (4 mL),

and the resulting mixture was stirred at 708C for 18 h. The solvent

was evaporated in vacuo, and the residue was purified by prepara-

tive HPLC (see general comments for details), affording compound

35 as an orange oil (85 mg, 65%): ¹H NMR (400 MHz, CDCl₃): d=

8.98 (brs, 1H), 8.50 (s, 1H), 8.43–8.34 (m, 2H), 8.14 (d, J=8.0 Hz,

2H), 7.34–7.21 (m, 4H), 7.12 (d, J=7.4 Hz, 1H), 4.26–4.18 (m, 4H),

3.56 (s, 2H), 3.32 (s, 3H), 2.62 (s, 3H), 2.07 ppm (s, 3H); LC/MS: m/

z: 526 [M+H]⁺; HPLC (method C): t_R=3.74 min (purity: 99.4%).

N-(2-Fluoro-4-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzyl)-N-methylglycine, hydrochloride salt

(33): Compound 33 was prepared following the procedure de-

scribed for the preparation of compound 28, replacing amidoxime

52k with 52l. It was isolated as a white powder (172 mg, 74%):

¹H NMR (300 MHz, [D₆]DMSO): d=8.34 (d, J=1.4 Hz, 1H), 8.18 (dd,

J=7.9, 1.7 Hz, 1H), 8.08 (dd, J=7.9, 1.3 Hz, 1H), 7.99 (dd, J=10.2,

1.3 Hz, 1H), 7.91 (t, J=7.7 Hz, 1H), 7.44 (d, J=7.9 Hz, 1H), 7.39–

7.27 (m, 3H), 7.15 (d, J=7.0 Hz, 1H), 4.50 (s, 2H), 4.26–4.11 (m, 4H),

3.26 (s, 3H), 2.81 (s, 3H), 2.04 ppm (s, 3H); UHPLC/MS: m/z: 474.2

[MꢀH]^ꢀ, 476.0 [M+H]⁺; HPLC (method A): t_R=4.06 min (purity:

99.6%).

2-((4-(5-(2-(Methoxymethyl)-2’-methylbiphenyl-4-yl)-1,2,4-oxa-

diazol-3-yl)-3-(trifluoromethyl)benzyl)(methyl)amino)acetic acid

(36): Compound 36 was prepared following the procedure de-

scribed for the preparation of compound 35, replacing amidoxime

52n with 52o. It was isolated as a pale yellow oil (77 mg, 60%):

¹H NMR (400 MHz, [D₆]DMSO): d=8.31 (d, J=1.8 Hz, 1H), 8.14 (dd,

J=7.9, 1.9 Hz, 1H), 8.00 (s, 1H), 7.95 (d, J=7.9 Hz, 1H), 7.85 (d, J=

8.0 Hz, 1H), 7.39 (d, J=7.9 Hz, 1H), 7.35–7.25 (m, 3H), 7.12 (d, J=

7.4 Hz, 1H), 4.27–4.11 (m, 2H), 3.90 (s, 2H), 3.34 (s, 2H), 3.23 (s,

2-((3-Fluoro-4-(5-(2-(methoxymethyl)-2’-methylbiphenyl-4-yl)-

1,2,4-oxadiazol-3-yl)benzyl)(methyl)amino)acetic acid, hydro-

chloride salt (34): Step 1: Using the procedure for compound 28,

replacing amidoxime 52k with 52m, (3-fluoro-4-(5-(2-(methoxy-

&

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3H), 2.35 (s, 3H), 2.03 ppm (s, 3H); LC/MS: m/z: 526 [M+H]⁺; HPLC

(method C): t_R=3.28 min (purity: 99.8%).

HPLC (method A): t_R=4.16 min (purity: 99.5%); Anal. calcd for

C₂₇H₂₆N₃O₄F·HCl: C 63.34, H 5.32, N 8.21, Cl 6.92, found: C 62.95, H

5.24, N 8.23, Cl 6.67.

2-(((5-(5-(2-(Methoxymethyl)-2’-methylbiphenyl-4-yl)-1,2,4-oxa-

diazol-3-yl)pyridin-2-yl)methyl)(methyl)amino)acetic acid (37):

Step 1: Using the procedure described for the preparation of com-

pound 35 (step 1), replacing amidoxime 52n with 52p, and follow-

ing purification by flash chromatography (iso-hexane/EtOAc, 1:1),

tert-butyl N-[(5-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-1,2,4-

oxadiazol-3-yl}pyridin-2-yl)methyl]-N-methylglycinate was isolated

({4-Fluoro-3-[5-(2-methoxymethyl-2’-methyl-biphenyl-4-yl)-

[1,2,4]oxadiazol-3-yl]-benzyl}methylamino)acetic acid (41): Com-

pound 41 was prepared following the procedure described for the

preparation of compound 3, using carboxylic acid 56a and ami-

doxime 52t. It was isolated as a white solid (185 mg, 87%):

¹H NMR (300 MHz, [D₆]DMSO): d=8.39 (dd, J=6.8, 2.3 Hz, 1H), 8.34

(d, J=1.7 Hz, 1H), 8.18 (dd, J=7.9, 1.9 Hz, 1H), 7.89–7.78 (m, 1H),

7.62 (dd, J=10.5, 8.5 Hz, 1H), 7.46 (d, J=8.0 Hz, 1H), 7.41–7.25 (m,

3H), 7.20–7.10 (m, 1H), 4.46 (s, 2H), 4.30–4.14 (m, 2H), 4.09 (s, 2H),

3.26 (s, 3H), 2.80 (s, 3H), 2.04 ppm (s, 3H); UHPLC/MS: m/z: 474.2

[MꢀH]^ꢀ, 476.0 [M+H]⁺; HPLC (method A): t_R=3.98 min (purity:

98.0%).

1

as an off-white solid (550 mg, 42%): H NMR (400 MHz, CDCl₃): d=

9.35–9.34 (m, 1H), 8.46–8.43 (m, 2H), 8.19–8.17 (m, 1H), 7.71–7.68

(m, 1H), 7.36–7.28 (m, 4H), 7.14–7.12 (m, 1H), 4.27–4.20 (m, 2H),

3.95 (s, 2H), 3.33 (s, 3H), 3.21 (s, 2H), 2.46 (s, 3H), 2.05 (s, 3H),

1.50 ppm (s, 9H).

Step 2: tert-Butyl N-[(5-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-

yl]-1,2,4-oxadiazol-3-yl}pyridin-2-yl)methyl]-N-methylglycinate

(0.550 g, 1.07 mmol) was dissolved in 4m HCl in dioxane (15 mL),

and the reaction mixture was stirred at RT for 2 h and then at 808C

for 1 h. The reaction mixture was allowed to cool and the suspen-

sion was filtered. The solid was washed with Et₂O (10 mL) and

dried in vacuo. The crude material was triturated with CH₂Cl₂to

({3-[5-(2-Methoxymethyl-2’-methyl-biphenyl-4-yl)-[1,2,4]oxadia-

zol-3-yl]-5-trifluoromethylbenzyl}methylamino)acetic acid (42):

Compound 42 was prepared following the procedure described

for the preparation of compound 3, using carboxylic acid 56a and

amidoxime 52u. It was isolated as a white solid (225 mg, 83%):

¹H NMR (300 MHz, [D₆]DMSO): d=8.62 (s, 1H), 8.48–8.32 (m, 2H),

8.21 (dt, J=4.1, 2.0 Hz, 2H), 7.45 (d, J=7.9 Hz, 1H), 7.41–7.23 (m,

3H), 7.15 (d, J=7.2 Hz, 1H), 4.53 (s, 2H), 4.32–4.14 (m, 2H), 4.08 (s,

2H), 3.26 (s, 3H), 2.79 (s, 3H), 2.04 ppm (s, 3H); UHPLC/MS: m/z:

524.1 [MꢀH]^ꢀ, 526.0 [M+H]⁺; HPLC (method A): t_R=5.03 min

(purity: 99.5%).

1

afford compound 37 as an off-white solid (0.420 g, 74%): H NMR

(400 MHz, [D₆]DMSO/D₂O): d=9.31 (s, 1H), 8.57 (dd, J=8.2, 2.3 Hz,

1H), 8.30 (d, J=2.3 Hz, 1H), 8.19–8.16 (m, 1H), 7.77 (d, J=7.6 Hz,

1H), 7.42–7.26 (m, 4H), 7.11–7.10 (m, 1H), 4.65 (s, 2H), 4.26–4.12

(m, 4H), 3.21 (s, 3H), 2.92 (s, 3H), 2.00 ppm (s, 3H); LC/MS: m/z:

459 [M+H]⁺; HPLC (method C): t_R=8.60 min (purity: 98.5%).

({2-Fluoro-3-[5-(2-methoxymethyl-2’-methyl-biphenyl-4-yl)-

[1,2,4]oxadiazol-3-yl]-benzyl}methylamino)acetic acid, hydro-

chloride salt (38): Compound 38 was prepared following the pro-

cedure described for the preparation of compound 3, using car-

boxylic acid 56a and amidoxime 52q. It was isolated as a white

({4-[5-(2-Ethoxy-2’-methyl-biphenyl-4-yl)-[1,2,4]oxadiazol-3-yl]-

benzyl}methylamino)acetic acid (43): Compound 43 was pre-

pared following the procedure described for the preparation of

compound 28, using carboxylic acid 56c and amidoxime 52 f. It

was isolated as a white solid (157 mg, 83%): ¹H NMR (300 MHz,

[D₆]DMSO): d=8.21 (d, J=8.3 Hz, 2H), 7.86 (dd, J=7.9, 1.4 Hz, 1H),

7.82–7.76 (m, 3H), 7.41 (d, J=7.8 Hz, 1H), 7.33–7.23 (m, 3H), 7.17

(d, J=6.8 Hz, 1H), 4.44 (s, 2H), 4.19 (q, J=7.0 Hz, 2H), 4.08 (s, 2H),

2.80 (s, 3H), 2.12 (s, 3H), 1.25 ppm (t, J=6.9 Hz, 3H); UHPLC/MS:

m/z: 456.1 [MꢀH]^ꢀ, 458.0 [M+H]⁺; HPLC (method A): t_R=4.17 min

(purity: 99.4%).

1

solid (140 mg, 86%): H NMR (300 MHz, [D₆]DMSO): d=8.34 (d, J=

1.7 Hz, 1H), 8.32–8.24 (m, 1H), 8.18 (dd, J=7.9, 1.9 Hz, 1H), 7.94–

7.85 (m, 1H), 7.57 (t, J=7.8 Hz, 1H), 7.45 (d, J=7.9 Hz, 1H), 7.40–

7.25 (m, 3H), 7.15 (d, J=7.2 Hz, 1H), 4.52 (s, 2H), 4.31–4.03 (m,

4H), 3.26 (s, 3H), 2.81 (s, 3H), 2.04 ppm (s, 3H); LC/MS: m/z: 476.2

[M+H]⁺, 474.3 [MꢀH]^ꢀ; HPLC (method A): t_R=3.96 min (purity:

97.6%).

N-(4-{5-[2-(Methoxymethyl)-2’-(trifluoromethyl)biphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzyl)-N-methylglycine, hydrochloride salt

(44): Compound 44 was prepared following the procedure de-

scribed for the preparation of compound 3, using carboxylic acid

56b and amidoxime 52 f. It was isolated as an off-white solid

(353 mg, 73%): ¹H NMR (300 MHz, [D₆]DMSO): d=8.32 (d, J=

1.7 Hz, 1H), 8.26–8.12 (m, 3H), 7.96–7.87 (m, 1H), 7.86–7.64 (m,

4H), 7.45 (dd, J=12.4, 7.9 Hz, 2H), 4.49 (s, 2H), 4.28–4.02 (m, 4H),

3.24 (s, 3H), 2.82 ppm (s, 3H); UHPLC/MS: m/z: 512.0 [M+H]⁺,

510.0 [MꢀH]^ꢀ; HPLC (method A): t_R=4.04 min (purity: 98.8%).

N-(2-Fluoro-5-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzyl)-N-methylglycine, hydrochloride salt

(39): Compound 39 was prepared following the procedure de-

scribed for the preparation of compound 8, using carboxylic acid

56a and amidoxime 52r. The solvents were evaporated to afford

compound 39 as

a

white powder (512 mg, 67%): ¹H NMR

(300 MHz, [D₆]DMSO): d=8.46 (dd, J=7.0, 2.1 Hz, 1H), 8.33 (d, J=

1.5 Hz, 1H), 8.30–8.26 (m, 1H), 8.17 (dd, J=7.9, 1.9 Hz, 1H), 7.59 (t,

J=9.2 Hz, 1H), 7.45 (d, J=8.0 Hz, 1H), 7.38–7.27 (m, 3H), 7.15 (d,

J=7.0 Hz, 1H), 4.55–4.51 (m, 2H), 4.26–4.16 (m, 4H), 3.26 (s, 3H),

2.83 (s, 3H), 2.04 ppm (s, 3H); UHPLC/MS: m/z: 474.3 [MꢀH]^ꢀ,

476.2 [M+H]⁺; HPLC (method A): t_R=4.54 min (purity: 98.4%).

N-Methyl-N-(4-{5-[2-methyl-2’-(trifluoromethyl)biphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzyl)glycine, hydrochloride salt (45): Com-

pound 45 was prepared following the procedure described for the

preparation of compound 3, using carboxylic acid 56h and ami-

doxime 52 f. It was isolated as a white solid (433 mg, 75%):

¹H NMR (300 MHz, [D₆]DMSO): d=8.25–8.16 (m, 3H), 8.12–8.05 (m,

1H), 7.91 (d, J=7.2 Hz, 1H), 7.84–7.76 (m, 3H), 7.74–7.65 (m, 1H),

7.47–7.37 (m, 2H), 4.47 (s, 2H), 4.11 (s, 2H), 2.81 (s, 3H), 2.12 ppm

(s, 3H); UHPLC/MS: m/z: 480.1 [MꢀH]^ꢀ, 482.0 [M+H]⁺; HPLC

(method A): t_R=4.18 min (purity: 99.8%); Anal. calcd for

C₂₆H₂₂N₃O₃F₃·HCl: C 60.29, H 4.48, N 8.11, Cl 6.85, found: C 60.14, H

4.36, N 8.12, Cl 6.81.

N-(3-Fluoro-5-{5-[2-(methoxymethyl)-2’-methylbiphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzyl)-N-methylglycine, hydrochloride salt

(40): Compound 40 was prepared following the procedure de-

scribed for the preparation of compound 3, using carboxylic acid

56a and amidoxime 52s. It was isolated as a white solid (153 mg,

76%): ¹H NMR (300 MHz, [D₆]DMSO): d=8.34 (d, J=1.8 Hz, 1H),

8.21–8.12 (m, 2H), 8.03–7.93 (m, 1H), 7.78–7.68 (m, 1H), 7.44 (d,

J=8.0 Hz, 1H), 7.38–7.25 (m, 3H), 7.14 (d, J=7.3 Hz, 1H), 4.48 (s,

2H), 4.31–4.08 (m, 2H), 4.10 (s, 2H), 3.25 (s, 3H), 2.81 (s, 3H),

2.04 ppm (s, 3H); UHPLC/MS: m/z: 474.3 [MꢀH]^ꢀ, 476.2 [M+H]⁺;

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N-{4-[5-(2,2’-Dimethylbiphenyl-4-yl)-1,2,4-oxadiazol-3-yl]benzyl}-

N-methylglycine, hydrochloride salt (46): Compound 46 was pre-

pared following the procedure described for the preparation of

compound 3, using carboxylic acid 56d and amidoxime 52 f. It

was isolated as a white solid (187 mg, 44%): ¹H NMR (300 MHz,

[D₆]DMSO): d=8.20–8.11 (m, 3H), 8.07–8.02 (m, 1H), 7.80 (d, J=

7.8 Hz, 2H), 7.41–7.26 (m, 4H), 7.13–7.10 (m, 1H), 4.48 (brs, 2H),

4.11 (brs, 2H), 2.81 (s, 3H), 2.14 (s, 3H), 2.04 ppm (s, 3H); UHPLC/

MS: m/z: 428.3 [M+H]⁺, 426.4 [MꢀH]^ꢀ; HPLC (method A): t_R=

4.14 min (purity: 100%).

ing studies by nitrogen cavitation. [³³P]Sphingosine-1-phosphate

(3000 Cimmol^ꢀ1

; American Radiolabeled Chemicals, Inc.) was

added to test compound in 2% final DMSO by competition. Crude

membrane fractions and wheat germ agglutinin (WGA) scintillation

proximity assay (SPA) beads (GE Healthcare) were added to give

a

final assay concentrations of 30 pm, 15 pm, 25 pm

[³³P]sphingosine-1-phosphate (for S1P_1,3,5, respectively), 50 mm 4-(2-

hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.5,

5 mm MgCl₂, 100 mm NaCl, 0.4% fatty acid-free bovine serum albu-

min (BSA), 1–5 mg/well of proteins and 100 mg/well of WGA SPA

beads in 96-well format. Filter binding was performed in an assay

mixture containing 50 mm HEPES, pH 7.5, 5 mm MgCl₂, 100 mm

NaCl, 0.4% fatty acid-free BSA, 6 mg/well of S1P₄membranes and

100 pm [³³P]sphingosine-1-phosphate in 96-well plates. Binding

was performed for 60 min and 90 min for S1P_1,3,5and S1P₄, respec-

tively, at RT on a shaker. Bound radioactivity was measured on

a PerkinElmer 1450 MicroBeta counter for S1P_1,3,5, and the mixture

was filtered by vacuum through a Whatman GF/C filter for S1P₄.

The filter was washed, and radioactivity was then determined in

scintillation mix cocktail on a PerkinElmer 1450 MicroBeta counter.

Triplicate samples were averaged and normalized as percentage of

inhibition relative to total binding (DMSO control wells) and non-

specific binding (1000-fold excess of unlabeled S1P for S1P_1,3,5and

phytosphingosine phosphate for S1P₄). Ten-point dose–response

curves allowed IC₅₀determination for each compound. K_ivalues

were calculated according to the Cheng–Prusoff equation. Each re-

ported K_ivalue is the average of a minimum of two independent

experiments, unless indicated otherwise.

N-Methyl-N-(4-{5-[2’-methyl-2-(trifluoromethyl)biphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzyl)glycine, hydrochloride salt (47): Com-

pound 47 was prepared following the procedure described for the

preparation of compound 28, using carboxylic acid 56 f and ami-

doxime 52 f. It was isolated as a white solid (172 mg, 85%):

¹H NMR (300 MHz, [D₆]DMSO): d=8.55 (s, 1H), 8.51 (d, J=8.0 Hz,

1H), 8.23 (d, J=8.1 Hz, 2H), 7.80 (d, J=8.1 Hz, 2H), 7.68 (d, J=

7.9 Hz, 1H), 7.42–7.26 (m, 3H), 7.17 (d, J=7.3 Hz, 1H), 4.46 (s, 2H),

4.10 (s, 2H), 2.80 (s, 3H), 2.03 ppm (s, 3H); UHPLC/MS: m/z: 480.1

[MꢀH]^ꢀ, 482.0 [M+H]⁺; HPLC (method A): t_R=4.76 min (purity:

100.0%).

N-(2-Fluoro-4-{5-[2’-methyl-2-(trifluoromethyl)biphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzyl)-N-methylglycine, hydrochloride salt

(48): Compound 48 was prepared following the procedure de-

scribed for the preparation of compound 28, using carboxylic acid

56 f and amidoxime 52l. It was isolated as a white solid (192 mg,

86%): m.p.: 1838C; ¹H NMR (300 MHz, [D₆]DMSO): d=8.56 (d, J=

1.3 Hz, 1H), 8.51 (dd, J=7.9, 1.5 Hz, 1H), 8.09 (dd, J=8.0, 1.5 Hz,

1H), 8.02 (dd, J=10.2, 1.3 Hz, 1H), 7.91 (t, J=7.7 Hz, 1H), 7.69 (d,

J=8.0 Hz, 1H), 7.42–7.26 (m, 3H), 7.17 (d, J=7.5 Hz, 1H), 4.50 (s,

2H), 4.13 (s, 2H), 2.81 (s, 3H), 2.03 ppm (s, 3H); UHPLC/MS: m/z:

498.1 [MꢀH]^ꢀ, 500.0 [M+H]⁺; HPLC (method A): t_R=4.35 min

(purity: 99.9%); Anal. calcd for C₂₆H₂₁N₃O₃F₄·HCl·H₂O: C 56.38, H

4.37, N 7.59, Cl 6.40, found: C 56.18, H 4.29, N 7.51, Cl 6.12.

Microsomal stability: The stability of the test compounds was stud-

ied in vitro in mouse and human liver microsomes. Microsomes

(final concentration 0.5 mgmL^ꢀ1), 50 mm phosphate buffer

(pH 7.4), NADPH (final concentration 1.5 mm) and compound (final

concentration 1 mm) were added to the assay plate. The microsome

suspension was added to initiate the reaction, and the plate was

incubated at 378C. The reaction was stopped by addition of cold

acetonitrile at the appropriate time points (t= 0, 5, 15 and 45 min).

The samples were centrifuged at 4000 rpm for 30 min at 48C and

analyzed by LC-MS/MS. The in vitro intrinsic clearance (Cl_int) was

calculated from the rate of compound disappearance.

N-Methyl-N-(3-{5-[2’-methyl-2-(trifluoromethyl)biphenyl-4-yl]-

1,2,4-oxadiazol-3-yl}benzyl)glycine (49): Compound 49 was pre-

pared following the procedure described for the preparation of

compound 8, using carboxylic acid 56 f and amidoxime 52i. It was

isolated as

a

white solid (321 mg, 90%): ¹H NMR (300 MHz,

[D₆]DMSO): d=8.55 (s, 1H), 8.50 (d, J=8.0 Hz, 1H), 8.36 (s, 1H),

8.23 (d, J=7.8 Hz, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.78–7.63 (m, 2H),

7.42–7.12 (m, 3H), 7.17 (d, J=7.4 Hz, 1H), 4.51 (s, 2H), 4.12 (s, 2H),

2.81 (s, 3H), 2.02 ppm (s, 3H); HPLC (method A): t_R=4.20 min

(purity: 99.48%); Anal. calcd for C₂₆H₂₂O₃F₃N₃·HCl: C 60.29, H 4.48,

N 8.11, found: C 60.08, H 4.42, N 7.91.

Cytochrome P450 (CYP) inhibition: Seven human recombinant CYP

isoenzymes were tested (1A2, 2C9, 2C19, 2C8, 2B6, 2D6 and 3A4).

The aim of this method is to determine the concentration of a com-

pound required to obtain 50% inhibition of the recombinant

human CYP. The assay is based on the Promega P450-Glo screen-

ing system, which includes a luminogenic substrate, an NADPH-re-

generating system and a luciferin detection reagent. CYP activity is

measured by convertion of the substrate (e.g., luciferin-ME EGE for

CYP 2B6) to luciferin EGE, which is further derivatised to d-luciferin

(readout via light emission using luciferin detection reagent). The

CYP membranes were prepared from baculovirus-infected insect

cells and contain human CYP and CYP reductase. In order not to

be depleted in NADPH cofactor during the course of the reaction,

the assay includes a NADPH regeneration system. Test compounds

were pre-incubated for 15 min at RT with the CYP enzyme (and ap-

propriate cofactors), in the absence of substrate. Then the enzy-

matic reaction was initiated by the addition of the substrate, fol-

lowed by 30 min of incubation at 378C. As controls, blank and neu-

tral values were tested on each plate. In the neutral control, DMSO

was added in place of the test compound; it defines the maximum

achievable substrate conversion, reflected by the strongest lumi-

nescence signal. In the blank control, a membrane fraction devoid

of CYP activity (control membranes prepared from wild-type bacu-

{3-[5-(2’-Methyl-2-trifluoromethyl-biphenyl-4-yl)-[1,2,4]oxadiazol-

3-yl]-benzyloxy}-acetic acid (50): Compound 50 was prepared fol-

lowing the procedure described for the preparation of compound

3, using carboxylic acid 56 f and amidoxime 52d. It was isolated as

a white solid (140 mg, 65%): ¹H NMR (300 MHz, [D₆]DMSO): d=

12.78 (brs, 1H), 8.55 (d, J=1.4 Hz, 1H), 8.51 (dd, J=7.8, 1.6 Hz,

1H), 8.14 (s, 1H), 8.11–8.05 (m, 1H), 7.66 (d, J=7.9 Hz, 1H), 7.63 (s,

1H), 7.61 (s, 1H), 7.42–7.26 (m, 3H), 7.18 (d, J=7.5 Hz, 1H), 4.69 (s,

2H), 4.15 (s, 2H), 2.03 ppm (s, 3H); UHPLC/MS: m/z: 467.1 [MꢀH]^ꢀ,

468.9 [M+NH₄]⁺; HPLC (method A): t_R=5.45 min (purity: 100%).

In vitro assays

Binding assays: Clonal Chinese hamster ovary (CHO) K1 cells ex-

pressing high levels of S1P_1,3,4,5receptors were isolated, and crude

membranes from these cells were prepared for use in ligand bind-

&

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24

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ÝÝ These are not the final page numbers!

Full Papers

lovirus-infected cells), substitutes the membranes from CYP-ex-

pressing cells, consequently, no substrate conversion is observed.

The amount of control membranes in blank controls was calculat-

ed to match the amount of membrane proteins present in the

CYP-positive membranes used in the rest of the assay plate. The

measured signal in blank wells describes the background value.

The % inhibition was determined for each inhibitor concentration,

and the corresponding IC₅₀value was calculated by nonlinear

curve fitting. The IC₅₀value was determined in triplicate, using ten

different concentrations of test compound. As an additional con-

trol, the IC₅₀value of a well-characterized CYP inhibitor (reference

control) was determined for each isoform.

Merck Serono, and approved the General Health Direction of the

Republic and Canton of Geneva, Animal Experimentation Domain

(Republique et Canton de Genꢄve, Direction Gꢅnꢅrale de la Santꢅ,

Domaine de l’expꢅrimentation animale), under the license numbers

1040/3338/1-2R and 1040/3602/3. Mice were acquired from Elev-

age Janvier (Le Genest-Saint-Isle, France) and were acclimatized for

at least one week prior to experimentation. During the acclimatiza-

tion period and the entire length of the studies, mice were main-

tained at 12/12 h light/dark cycle and given food and water ad libi-

tum.

Unbound fraction in the brain: Individual na¨ıve mouse brains were

harvested from female mice purchased from Janvier. Brains were

homogenized in phosphate-buffered saline (PBS) (brain/PBS, 1:2

w/v) using a Precellys 24 tissue homogenizer. Homogenates were

stored in aliquots at ꢀ208C until use. Membranes (3.5 kDa cut-off)

were conditioned in deionized water for 60 min, followed by con-

ditioning in water/EtOH (8:2) for 30 min, and rinsed in deionized

water before use. On the donor side, diluted matrices were spiked

with the test compound (~500 mgmL^ꢀ1for brain homogenate),

and 150 mL aliquots (n=6 replicates for each test compound) were

loaded into the upper-half-well of the equilibrium dialysis plate.

On the acceptor side, PBS (150 mL) was loaded in the lower-half-

well of the equilibrium dialysis plate. Dialysis versus PBS was car-

ried out for 5 h at 378C in a temperature-controlled incubator

using a microplate shaker. After 5 h, on the donor side, matrix ali-

quots (10 mL) were transferred to an Eppendorf tube containing

100 mL of 0.5 mgmL^ꢀ1of internal standard (IS) in CH₃CN. Fresh PBS

was added to each eppendorf tube (50 mL) and vortex mixed. On

the acceptor side, PBS aliquots (50 mL) were transferred to an Ep-

pendorf tube containing 100 mL of 0.5 mgmL^ꢀ1of ISTD in CH₃CN.

Na¨ıve mouse brain was added to each Eppendorf tube (10 mL) and

vortex mixed. Samples were then centrifuged for 15 min at

4000 rpm at 48C. Supernatant (100 mL) was recovered in vials and

analyzed by LC-MS/MS. The unbound fraction was calculated as

described by Kalvass and Maurer.^[35]

Protein binding assays: Protein binding of test compounds was de-

termined by ultrafiltration using serum from various species

(mouse, rat, human, etc.). Test compound (final concentration

5 mm) was incubated in triplicate at three different serum dilutions

(1:2, 1:5 and 1:10) for 30 min at 378C using slight agitation. After

incubation, the 96-well filter plates were centrifuged for 45 min at

3500 rpm and 378C. Filtrate samples (25 mL) were treated with

EtOH (50 mL) and an internal standard solution (50 mL), and ana-

lyzed by LC-MS/MS. The fraction unbound was calculated from the

drug concentrations in the filtrate samples.

hERG patchclamp assay:^[36]Samples of test compounds and posi-

tive controls were prepared fresh daily by diluting stock solutions

into HEPES-buffered physiological saline (HB-PS) solution (NaCl,

137 mm; KCl, 4.0 mm; CaCl₂, 1.8 mm; MgCl₂, 1 mm; HEPES, 10 mm;

glucose, 10 mm), and the pH was adjusted to 7.4 with aq NaOH

(5n). HB-PS was prepared weekly and refrigerated until use. Since

previous results have shown that ꢃ0.3% DMSO does not affect

channel current, all test and control solutions contained 0.3%

DMSO. Each test compound formulation was sonicated at RT for at

least 20 min to facilitate dissolution. A glass-lined 96-well com-

pound plate was loaded with the appropriate amounts of test and

control solutions, and placed in the plate slot of a QPatch HT

system (Sophion Bioscience A/S, Denmark). In this study, hERG

channels were expressed in a CHO cell line that lacks endogenous

rapidly activating delayed rectifier (I_Kr) channel. CHO cells were

stably transfected with hERG cDNA. Stable transfectants were se-

lected by coexpression with an antibiotic-resistance gene incorpo-

rated into the expression plasmid. Selection pressure was main-

tained by including the selection antibiotic in the culture medium.

Cells were cultured in nutrient mixture F-12 (F-12) supplemented

with 10% fetal bovine serum, 100U mL^ꢀ1penicillin G sodium,

100 mgmL^ꢀ1streptomycin sulfate. Before testing, cells in culture

dishes were washed twice with Hank’s balanced salt solution, treat-

ed with accutase and resuspended in the CHO serum-free media

(1–1.5ꢂ10⁶cellsmL^ꢀ1). Intracellular solution for whole-cell record-

ings consisted of potassium aspartate (130 mm), MgCl₂(5 mm), eth-

ylene glycol tetraacetic acid (EGTA) (5 mm), ATP (4 mm), HEPES

(10 mm), and the pH was adjusted to 7.2 with aq KOH (5n). An in-

tracellular solution was loaded into the intracellular compartments

of the QPlate. Cell suspension was pipetted into the extracellular

compartments of the QPlate. After establishment of a whole-cell

configuration, membrane currents were recorded using the QPatch

HT system. Before digitization, the current records were low-pass

filtered at one-fifth of the sampling frequency.

Model of S1P receptor agonist-induced lymphopenia in mice: Female

C57BL/6 mice (8 week old) received S1P receptor agonists by oral

route using 0.5% carboxymethylcellulose (CMC)/0.25% Tween20 in

water as a vehicle. Lymphopenia was measured at 24, 48 and 72 h

after the administration of test compound. For that purpose, blood

was sampled via intracardiac puncture under isoflurane anesthesia.

Mice received an intraperitoneal (ip) injection of 100 IUkg^ꢀ1hepa-

rin (liquemin, Roche) 15 min prior to blood sampling. Total and dif-

ferential blood cell counts were performed (including lymphocyte

counts used for assessing lymphopenia) using a Beckman/Coulter

counter (ACT5Diff AD32097). The quality of blood sampling was

confirmed by assessing erythocytes and platelets counts. The PK

profiles were determined from this experiment. After counting, the

blood was centrifuged (6000 rpm, 10 min, 48C) and plasma (50 mL/

mouse/time point) and brain sampling were performed at 2 h (n=

3 mice) and 24, 48 h (n=8 mice each) post-dose at sacrifice.

Lymph nodes were collected at 24, 48 and 72 h post-dose at sacri-

fice. Plasma, brain and lymph nodes were stored at ꢀ208C until

analysis. For bioanalysis, samples were processed by protein pre-

cipitation and analyzed using LC-MS/MS.

MOG_35ꢀ55-induced experimental autoimmune encephalomyelitis (EAE)

in mice: EAE was induced in 9–11 week-old female C57BL/6 mice

by immunization against rat myelin oligodendrocyte glycoprotein

peptide (rMOG_35–55). Each mouse received a dose of pertussis toxin

(Alexis, cat# 630-003-C050, 300 ng/mouse in 200 mL PBS) adminis-

tered ip and 100 mL of an emulsion containing rMOG_35ꢀ55(200 mg/

mouse; NeoMPS, cat# SC1272), Mycobacterium tuberculosis

In vivo biological methods

Animal welfare: All experimental protocols involving animals were

performed in accordance with international standards for animal

experimentation, reviewed by an internal ethics committee at

ChemMedChem 0000, 00, 0 – 0

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25

ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

&

These are not the final page numbers! ÞÞ

Full Papers

mann, M. Traebert, C. Bruns, S. Pan, N. S. Gray, K. Hinterding, N. G.

Cooke, A. Groenewegen, A. Vitaliti, T. Sing, O. Luttringer, J. Yang, A.

Gardin, N. Wang, W. J. Crumb Jr., M. Saltzman, M. Rosenberg, E. Wall-

strçm, Br. J. Pharmacol. 2012 , 167 , 103 5 –1 047.

(0.25 mg/mouse; DIFCO, cat# 231141) in complete Freund’s adju-

vant (DIFCO, cat# 263810) by subcutaneous injection into the

back. For sham-immunization, the rMOG_35ꢀ55solution used for the

preparation of the emulsion was replaced by PBS. Two days later,

the injection of Pertussis toxin was repeated. After EAE induction,

mice were weighed daily, and the neurological impairment was

quantified using a 15-point scale individually assessing the paraly-

sis of the tail (0=no motor impairment; 1=flaccid extremity of

the tail; 2=completely flaccid tail), of the hind limbs (0=no motor

impairment; 1=grid test positive, that is, mouse misplaces its

steps when allowed to walk on the cage grid; 2=flip test positive;

3=mouse cannot hold on to the underside of the wire cage top

for more than a few seconds using all paws; 4=complete parape-

sis (paralysis of lower limbs), of the fore limbs (0=no motor im-

pairment, 1=mild weakness of forelimbs; 2=one forelimb is com-

pletely paralyzed; 3=impossibility to move forward) and inconti-

nence (0=no incontinence; 1=incontinence). The final score for

each animal was determined by the addition of all the above-men-

tioned categories, therefore the maximum score for live animals

was 10. Death due to disease (or moribund mice that need to be

euthanized) was attributed a score of 15 for the day of the event.

At day 14 post-immunization (14 d.p.i.), mice were homogeneously

assigned into the different experimental groups according to the

clinical score and the body weight loss. Only mice that developed

clinical signs of disease were kept in the experiment. At this time

point, that is, at 14 d.p.i., the treatment with the test compound or

vehicle was initiated once daily five days per week. On the last day

of the experiment, 24 hous after the last administration, mice were

euthanized by CO₂overdose, and the blood was harvested for leu-

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AD32097) and plasma exposure determination.

Keywords: agonists · drug design · FTY-720 (fingolimod) ·

immunomodulators

· sphingosine-1-phosphate receptors ·

structure–activity relationships

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&

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ÝÝ These are not the final page numbers!

Full Papers

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pursued to avoid excessive lipophilicity. Meta- and para-substitution are

not expected to influence the dihedral angle sufficiently and were also

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19 ngmL^ꢀ1were measured with the doses of 1 and 3 mgkg^ꢀ1, respec-

tively. For compound 49, plasma exposures of 48ꢁ6 and 144ꢁ

8 ngmL^ꢀ1were measured with the doses of 1 and 3 mgkg^ꢀ1, respec-

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[28] CCDC 1037201 and 1037202 contain the supplementary crystallograph-

ic data for this paper. These data can be obtained free of charge from

the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/

data_request/cif.

[29] No activity against S1P₂was measured for S1P₁agonists disclosed in

Table 5. S1P₂activity was determined by cell-shape-induced impedance

changes caused by agonist stimulation of S1P₂-expressing Chinese

hamster ovary (CHO) cells (M. Jongsma, M. C. Hendriks-Balk, M. C.

Michel, S. L. Peters, A. E. Alewijnse, Br. J. Pharmacol. 2006 , 149 , 27 7 –

282) using Label-free CellKey technology.

Received: December 23, 2014

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Pharmacophore-based design of novel oxadiazoles as selective sphingosine-1-phosphate (S1P) receptor agonists with in vivo efficacy

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