Highly Selective and Potent Human β-Secretase 2 (BACE2) Inhibitors against Type 2 Diabetes: Design, Synthesis, X-ray Structure and Structure–Activity Relationship Studies

Accepted Article

Title: Highly Selective and Potent Human β-Secretase 2 (BACE2)

Inhibitors against Type 2 Diabetes: Design, Synthesis, X-ray

Structure and Structure-Activity Relationship Studies

Authors: Arun K Ghosh, Margherita Brindisi, Yu-Chen Yen, Emma

K. Lendy, Satish Kovela, Emilio Leal Cárdenas, Bhavanam

Sekhara Reddy, Kalapala Venketeswara Rao, Deborah

Downs, Xiangping Huang, Jordan Tang, and Andrew D.

Mesecar

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the VoR from the journal website shown below when it is published

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content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.201800725

Link to VoR: http://dx.doi.org/10.1002/cmdc.201800725

A Journal of

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Highly Selective and Potent Human β-Secretase 2 (BACE2) Inhibitors

against Type 2 Diabetes: Design, Synthesis, X-ray Structure and

Structure-Activity Relationship Studies

Arun K. Ghosh*^[a], Margherita Brindisi,^[a]Yu-Chen Yen,^[b]Emma K. Lendy,^[b]Satish Kovela,^[a]Emilio Leal

Cárdenas,^[a]Bhavanam Sekhara Reddy,^[a]Kalapala Venketeswara Rao,^[a]Deborah Downs,^[c]Xiangping

Huang,^[c]Jordan Tang,^[c]Andrew D. Mesecar^[b]

[a]

Prof. Dr. A. K. Ghosh, Dr. M. Brindisi, Dr. S. Kovela, Mr. E. L. Cárdenas, Dr. B. S. Reddy, and Dr. K. V. Rao

Department of Chemistry and Department of Medicinal Chemistry and Molecular Pharmacology

Purdue University, West Lafayette, IN 47907

E-mail: akghosh@purdue.edu

[b]

[c]

Prof. Dr. A. D. Mesecar, Dr. Y.-C. Yen, and Ms. E. K. Lendy

Department of Biochemistry

Purdue University, West Lafayette, IN 47907

Dr. J. Tang, Dr. X. Huang, Ms. D. Downs

Protein Studies Program

Oklahoma Medical Research Foundation

Department of Biochemistry and Molecular Biology

University of Okalahoma Health Science Center, Oklahoma City, OK 73104

Supporting information for this article is given via a link at the end of the document

Abstract: We herein present the design, synthesis, and biological

evaluation of potent and highly selective β-secretase 2 (memapsin 1,

beta-site amyloid precursor protein cleaving enzyme 2 or BACE 2)

inhibitors. BACE2 has been recognized as an exciting new target for

type 2 diabetes. The X-ray structure of BACE1 bound to inhibitor 2a

containing a hydroxyethylamine isostere was determined and based on

this structure, a computational docking study was performed which led

to inhibitor 2a-bound BACE2 models which were used to optimize the

potency and selectivity of inhibitors. A systematic structure-activity

relationship study led to identification of determinants of inhibitors’

potency and selectivity towards the BACE2 enzyme. Inhibitors 2d (K_i=

0.031 nM, selectivity over BACE1 174,000-fold) and 3l (K_i= 1.6 nM,

selectivity over BACE1 over 500-fold) displayed outstanding potency

and selectivity. Inhibitor 3l is non-peptide in nature and may pave the

way to the development of a new class of potent and selective BACE2

inhibitors with clinical potential.

Diabetes mellitus is a chronic, progressive metabolic

disorder. It is characterized by hyperglycemia resulting from

reduced insulin secretion from pancreatic beta-cells.⁶The disease

also originates from an increase in insulin demand associated with

tissue insulin resistance. There is no cure for type 2 diabetes,

however, treatment modalities include lifestyle modifications,

treatment of obesity, administration of oral hypoglycemic agents

and insulin sensitizers like metformin, a biguanide which reduces

insulin resistance, a recommended first-line medication particularly

for obese patients. Other effective antidiabetic medications include

sulfonylureas, meglitinide, thiazolidinediones, sodium-glucose co-

transporter 2 (SGLT2) inhibitors, and α-glucosidase inhibitors.^7-9

Moreover, enhancing incretin action has been, in recent years, a

widely utilized and effective approach for the treatment of type 2

diabetes. Strategies have been developed to circumvent dipeptidyl

peptidase 4 (DPP-4) action, leading to the development of distinct

glucagon-like peptide 1 (GLP-1) receptor agonists with longer half-

lives (such as exenatide) as well as compounds blocking DPP-4

action and leading to increased endogenous GLP-1 levels (eg.

sitagliptin).¹⁰Despite these currently available options for lowering

blood glucose, there are severe liabilities associated with these

Introduction

Type 2 diabetes mellitus is a major medical problem that

existing antidiabetic drugs, especially related to cardiovascular

seriously threatens human health worldwide, especially in

12

complications.^11,

Although examples of recent drugs with

2

developing countries.^1,

International Diabetes Federation

reduced risk of cardiovascular death have been reported (eg. the

SGLT2 inhibitor empagliflozin),¹³new therapies based upon

innovative mechanisms are still urgently needed to address

improvement of treatment and current unmet needs.

A recent study demonstrated that inhibition of BACE2 in

insulin-resistant mice resulted in an increase of beta-cell mass and

improvement of insulin levels in mice.¹⁴Importantly, another recent

study has shown that BACE2 suppression promotes beta-cell

survival and function in a model of type 2 diabetes induced by

human islet amyloid polypeptide over-expression.¹⁵Tmem27

estimated that worldwide, there were 415 million adults between

the age of 20-70 living with diabetes and 5 million deaths due to

diabetes in 2015.³The global burden for diabetes patient care

exceeded over 673 billion dollars in 2015.³The prevalence of

diabetes has been increasing steadily, particularly in developing

countries, with three quarters of patients being from low and middle

income families. In some countries, the number of patients affected

with diabetes is expected to increase even more rapidly due to the

aging population and adding further to the healthcare burden.^{4, 5}

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(collectrin) is a type 1 transmembrane protein and its over

expression in pancreatic beta-cells leads to cellular proliferation,

increased cell mass and increased insulin production. The excess

Tmem27 in beta-cells is regulated by BACE2 cleavage of

Tmem27, resulting in the release of the Tmem27 ectodomain

outside the cell.^16,17These studies suggested that BACE2

inhibitors may have the potential for novel treatments of type 2

diabetes.¹⁸

BACE2 (memapsin 1) is a type 1 membrane-bound aspartic

acid protease that belongs to the pepsin family.¹⁹It is a close

homologue of BACE1 which is highly expressed in the brain.^20,21

BACE2 is expressed in the alpha-cells of pancreatic islets and it

is found in other peripheral tissues, the colon and kidney.²²It

treatment of diabetes.^11,14,15However, BACE2 inhibitors may

need to be selective for a number of reasons. First of all, its

closest homologue, BACE1, has a distinct physiological function

in down-regulating brain neuronal function by cleaving APP and

overproducing β-amyloid peptide (Aβ) in the brain.²⁴While

BACE1 has been mechanistically an exciting target for possible

treatment of Alzheimer’s disease, BACE1 gene deletion resulted

in behavioral abnormalities in mice.²⁵Furthermore, BACE1 is

involved in the formation of myelin sheath in central and

peripheral nerves.^26,27BACE1 may also have other important

protein substrates.^26,28

Diabetes is a chronic disease and for effective chronic

treatment, any off-target toxicities need to be minimized, as

collective toxicity burden over time could be severe. Therefore,

development of selective BACE2 inhibitors over cathepsin D is

also critically important. Cathepsin D is abundant in cells and it

plays an important role in protein catabolism.^29,30In addition, it has

an important role in retinal function of the eyes.^31,32The majority

of current BACE1 inhibitors lack selectivity and as a result, clinical

toxicity has become a major issue. Thus far, there have been few

selective BACE1 inhibitors that have been taken through clinical

development.^33,34For the development of BACE2 inhibitor drugs

for diabetes, inhibitor selectivity is critically important and there

are only a few limited reports of BACE2 inhibitors with marginal

selectivity.^14,35We recently reported our preliminary work on the

design and synthesis of selective BACE2 inhibitors based upon

transition-state isosteres.³⁶We have now expanded structure-

activity relationship studies, determined a high-resolution X-ray

structure of a potent and selective inhibitor complexed with

BACE1 and created a structural model of inhibitor and BACE2

active site interactions. We herein report the full account of our

studies detailing the design, synthesis, biological evaluation, X-

ray crystallography and computational docking studies leading to

potent and selective BACE2 inhibitors.

Results and Discussion

In our preliminary efforts towards the design of potent and

selective BACE2 inhibitors, we focused our inhibitor design based

upon hydroxyethylamine isosteres as these dipeptide mimics are

inherent to several FDA approved HIV-1 protease inhibitor

drugs.^37,38Our starting point for the design of selective BACE2

inhibitors is compound 1 (Figure 1, K_i= 1.8 nM, BACE1) which

was designed by us as a potent BACE1 inhibitor.³⁹This inhibitor

exhibits many drug-like properties including low toxicity and

lipophilicity. Inhibitor 1 is quite potent against BACE1, but it also

displayed inhibition towards BACE2 with a K_ivalue of 137 nM

which is 76-fold less potent than BACE1.⁴⁰K_ivalues for these

compounds and all other compounds described herein were

determined from kinetic data determined under tight-binding

inhibition conditions and using the Morrison equation as

described previously.³⁶

Figure 1. Structures of BACE2 inhibitors 1, 2a, 2d and 3l.

We next determined a high-resolution X-ray structure of

inhibitor 1-bound BACE1 complex.³⁹The X-ray structure provided

critical ligand-binding site interactions in the BACE1 active site as

shown in Figure 2A. We have made a number of attempts to

crystallize inhibitor 1 in complex with BACE2 but so far we have

been unable to grow crystals suitable for structural studies. In the

absence of an X-ray structure of a compound 1-bound BACE2

has been demonstrated that BACE2 inhibition results in beta-cell

survival through a potential substrate, islet amyloid polypeptide

(IAPP). Overexpression of IAPP leads to defects in glucose

tolerance.²³BACE2 also cleaves plasma membrane protein

Tmem27 and regulates beta-cell mass and function.¹⁴Taken

together, BACE2 inhibition is potentially a viable new path for the

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Figure 2. (A) X-ray structure of inhibitor 1 (carbon chain, orange) bound to BACE1 (PDB code: 2VKM). (B) An energy-minimized model

of inhibitor 1 (carbon chain, green) bound in the BACE2 active site. All strong hydrogen bonding interactions are shown as dotted lines.

The docking was performed with AutoDock Vina (see Supplementary Material for details).

complex, we performed computational docking to create a model

of inhibitor 1 bound in the BACE2 active site. The model was

constructed using the available low-resolution X-ray structure of

an inhibitor-bound BACE2 complex.^41,42The resulting model of

inhibitor 1 bound in the active site of BACE2 is shown in Figure

2B. Our analysis of a compound 1-bound BACE2 computational

model and the X-ray structure of 1-bound BACE1, suggested that

certain BACE2 active site residues can be specifically targeted in

order to achieve potency and selectivity (e.g. Gly50 and Thr88).

Also, we hypothesized that the incorporation of polar substituents

on the prime side would reduce BACE1 activity thus leading to

more potent and selective BACE2 inhibitors. In particular, we

observed that the methoxybenzyl moiety in inhibitor 1 is mostly

engaged in non-polar interactions with residues in the

hydrophobic S1'-S2' subsites of BACE1. Therefore, replacement

of the methoxybenzyl group with polar functionalities could

improve selectivity towards BACE2. We therefore sought to

simultaneously insert alcohol and amide polar functionalities by

replacing the methoxybenzyl group with an allothreonine

isobutylamide and other derivatives.³⁶Thus, replacement of the

P1’-methoxybenzylamine of inhibitor 1 with an allothreonine

derivative resulted in inhibitor 2a. Further structural modification

led to the very potent and selective inhibitors 2d and 3l.

Chemistry

Our synthesis of BACE2 inhibitors 2f-j is shown in Scheme 1.

Various isophthalamide derivatives 4a-7b were prepared using

previously described procedures.^43-46For the synthesis of

allothreonine isobutylamide derivatives, the Boc group of known⁴³

isobutylamide derivatives 8a,b was deprotected in the presence

of trifluoroacetic acid (TFA) in CH₂Cl₂at 0 °C to 23 °C for 1 h.

Reaction of the resulting free amine with commercially available

tert-butyl ((S)-1-((S)-oxiran-2-yl)-2-phenylethyl) carbamate 9 in 2-

Scheme 1. Reagents and conditions (a) TFA, dry CH₂Cl₂, 1 h, from

propanol heated at 80 °C for 18 h, provided the corresponding

0 °C to 25 °C; (b). 9, i-PrOH, 80 °C, 18 h; (c) TFA, dry CH₂Cl₂, 1 h,

epoxide open product. Exposure of the resulting Boc derivative

from 0 °C to 25 °C, 66-86% (3 steps) (d) Suitable acids 5a,b and 6a,b

to TFA in CH₂Cl₂afforded amine derivatives 10a,b. Coupling of

acids 5a,b and 6a,b with amines 10a,b using EDCI and HOBt in

the presence of N,N-diisopropylethylamine (DIPEA) in CH₂Cl₂at

and appropriate amines 10a or 10b, EDCIꞏHCl, HOBtꞏH₂O, DIPEA,

dry CH₂Cl₂, 15 h, rt, 39-78%.

23 °C led to compounds 2f-j with yields ranging from 39% to 78%.

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The synthesis of aminoalcohol derivatives containing the

is shown in Scheme 3. Opening of epoxide 9 carried on with

amine derivatives 16a-c in isopropanol at 80 °C afforded

aminoalcohol derivatives 17a-c. Deprotection of Boc-group by

exposure to TFA followed by coupling of the resulting amines with

the suitable carboxylic acids 4a-d³⁶using EDCI and HOBt in the

presence of DIPEA led to final compounds 3a-h (Table 2).

substituted aromatic rings is shown in Scheme 2. Commercially

available aldehydes 11a,b were subjected to Wittig reaction with

the appropriate alkyl triphenylphosphonium bromide leading to

the corresponding olefins as E/Z mixture. Isomerization of the

olefin

mixture

in

the

presence

of

bis(acetonitrile)dichloropalladium(II) resulted in the E-olefins 12a-

c in nearly quantitative yields.⁴⁷Asymmetric dihydroxylation of

olefins 12a-c with AD-mix-α provided stereochemically defined

diols 13a-c. These diols were converted into the corresponding

diastereoisomeric mixtures (1:1) of cyclic sulfites 14a-c by

treatment with thionyl chloride in the presence of triethylamine.⁴⁸

Treatment of sulfites 14a-c with sodium azide in DMF at 80 °C,

afforded azidoalcohols 15a-c in excellent yields.

R²

CHO

a, b

R¹

, R¹= OMe, R²= Me

, R¹= OMe, R²= n-Pr

, R¹= H, R²= n-Pr

¹= OMe

12a

12b

12c

11a,

11b,

R

¹= H

c

R²

S

O

HO

OH

O

Scheme 3. Reagents and conditions. a) 16a, 16b or 16c, i-PrOH, 18

h, 80 °C, 65-71%; b) TFA, dry CH₂Cl₂, 2 h, from 0 °C to 25 °C; c) Acids

4a-d, EDCIꞏHCl, HOBtꞏH₂O, DIPEA, dry CH₂Cl₂, 15 h, 25 °C, 55-71%

(2 steps).

R²

d

O

R¹

, R¹= OMe, R²= Me

, R¹= OMe, R²= n-Pr

, R¹= H, R²= n-Pr

14a

14b

14c

13a

13b

13c

The syntheses of BACE2 inhibitors 3i-n (Table 3) with

variation of P3 and P2'-ligands of inhibitor 1 is shown in Scheme

4. Opening of epoxide 9 with various benzyl amine derivatives

provided amines 18a,b.^39,50These amines were coupled with

acids 4a, 4b,³⁶7a or 7b,⁵¹using EDCI and HOBt in the presence

of DIPEA to provide the final compounds 3i-n (Table 3). Inhibitor

20 was synthesized starting from acetophenone derivative 19

following reported procedures.³⁵

1

, R = OMe, R²= n-Pr

, R¹= H, R²= n-Pr

e

N₃

NH₂

R²

f, g

The synthesis of inhibitor 3o with a substituted oxazole

derivative as the P3-ligand is shown in Scheme 5. Alkylation of

commercially available oxazole 21 with methyl iodide in the

presence of sec-butillithium and TMEDA led to alkylated

derivative 22. Deprotection of the Boc group by exposure to TFA

followed by coupling of the resulting amine with acid 23 in the

presence of EDCI, HOBt and DIPEA furnished the amide

derivative. Hydrolysis of ester with aqueous LiOH led to acid

derivative 24. Coupling of acid 24 with amine 18b, provided final

inhibitor 3o in 48% yield.

OH

R¹

, R¹= OMe, R²= Me

, R¹= OMe, R²= n-Pr

, R¹= H, R²= n-Pr

, R¹= OMe, R²= Me

, R¹= OMe, R²= n-Pr

, R¹= H, R²= n-Pr

15a

16a

16b

16c

15b

15c

Scheme 2. Reagents and conditions. a) R²CH₂P⁺Ph₃Br^-, nBuLi, dry

THF, 1 h, -78 °C; b) (CH₃CN)₂Cl₂Pd, CHCl₃, 72 h, 25 °C, 69-84% (2

steps); c) AD-mix-α t-BuOH/H₂0, 24 h, 25 °C, 68-73%; d) TEA, thionyl

chloride, 0 °C, 45 min, 66-79%; e) NaN₃, DMF, 10 h, 80 °C, 74-88%;

f) (Boc)₂O, H₂, Pd/C, EtOAc, 1 atm, 25 °C, 15 h; g) TFA, dry CH₂Cl₂,

1 h, from 0 °C to 25 °C, 66-86% (2 steps).

Catalytic hydrogenation of these azides in the presence of di-t-

butyldicarbonate provided the Boc-protected aminoalcohols.

Exposure of these Boc-derivatives to TFA in CH₂Cl₂at 0 °C to

23 °C for 1 h furnished free aminoalcohols 16a-c.⁴⁹

The synthesis of BACE2 inhibitors 3a-h (Table 2) with

varying substituents on the aromatic rings and on the side chain

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Structure-Activity Relationships

At the outset, we replaced the methoxybenzylamine P1'-

ligand of inhibitor 1 with an allothreonine isobutyl amide group.

The resulting inhibitor 2a showed improvement of BACE2 potency

with a K_ivalue of 14.6 nM and demonstrated nearly 3-fold

selectivity over BACE1. We next determined the X-ray structure

of inhibitor 2a-bound BACE1 and created an active site model of

2a-bound to BACE2. The X-ray structure of 2a in complex with

BACE1 is shown in Figure 3 and the computational model of 2a

bound to the BACE2 active site is shown in Figure 4. We gained

critical molecular insights on specific active site interactions that

could improve potency and selectivity against the BACE2 enzyme.

In particular, from the model of compound 2a bound to the BACE2

enzyme, it appeared that the P2 amide NH and the sulfonamide

group on the P2-isophthalamide were not involved in significant

polar interactions, in contrast to interactions of 2a in BACE1 active

site. Based on this speculation, we first assessed compound 2b

as the amide N-methylated counterpart of compound 2a. The

resulting inhibitor 2b displayed reduction of BACE2 potency, but

improved selectivity over BACE1 (K_i= 80 nM for BACE2 and K_i=

1812 nM for BACE1).³⁶We then designed compound 2c in which

the sulfonamide moiety of 2a was replaced by a methyl group

(Compound 2c displayed a BACE1 K_ivalue of 731 nM and a

BACE2 K_ivalue of 183 nM). Our analysis of binding properties of

inhibitor 2c with respect to the model of compound 2a within the

BACE2 active site (Figure 4) suggested that a larger P1' alkyl

chain replacing the methyl group of allothreonine could result

more effective in establishing van der Waals interactions with the

surrounding hydrophobic residues.³⁶We also planned to alkylate

the amide NH and remove the P2-sulfonamide functionality. This

prompted us to design compounds 2d and 2e (Table 1), which

differ by the presence of an additional methyl on the isophthalic

moiety benzene ring. Both compounds displayed excellent

inhibitory activity on BACE2 (K_i= 0.031 nM for 2d and K_i= 0.038

nM for 2e) with inhibitor 2d showing outstanding selectivity over

BACE1 (174,000 fold).³⁶To date, the only other relevant work on

the design of selective BACE2 inhibitors is related to a patent

which reported a series of 2-aminodihydro[1,3]-thiazines, as

exemplified by inhibitor 20 (Scheme 4).³³

Scheme 4. Reagents and conditions. a) Acids 4a, 4b, 7a or 7b,

EDCIꞏHCl, HOBtꞏH₂O, DIPEA, dry CH₂Cl₂, 15 h, 25 °C, 55-71% (2

steps).

Based on the high potency of 2a against the purified BACE2

enzyme, we next investigated whether inhibitor 2a could prevent

processing of Tmem27 in the pancreatic beta-cell line MIN6.^52,53

Tmem27 behaves like a canonical BACE1/2 substrate, being

subjected to subsequent intramembrane cleavage by the -

secretase complex. This was confirmed by incubation of MIN6

cells with increasing concentrations of -secretase inhibitor N-[N-

(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester

(DAPT) which led to the stabilization of a 22 kDa C-terminal

fragment (CTF) in the cell lysate.¹⁴We tested our inhibitor 2a

following the protocol employed by Esterhazy.¹⁴Accordingly, the

C-terminal fragment of Tmem27 was preserved from degradation

in the presence of DAPT as shown in lane 1 (Figure 5). Without

the treatment of DAPT, most of the CTF of Tmem27 was

degraded as shown in lane 5. Hence, DAPT was added in the

cellular assay for studying the in cellulo potency of potent

Scheme 5. Reagents and conditions. a) s-BuLi, TMEDA, Et₂O, - 78 °C,

30 min, then MeI, -78 °C to 25 °C, 8 h, 86%; b) TFA:CH₂Cl₂(3:1),

25 °C, 1 h; c) 23, EDCI, HOBt, DIPEA, CH₂Cl₂, 0 °C to 25 °C, 12 h,

66% (2 steps); d) LiOH^.H₂O, THF:H₂O (2:1), 25 °C, 6 h, 88%; e) 18b,

EDCI, HOBt, DIPEA, CH₂Cl₂, 0 °C to 25 °C, 12 h, 48%.

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Figure 3. A Stereoview of the X-ray structure of inhibitor 2a (carbon chain, magenta color) in complex with BACE1 (memapsin 2)

(PDB code: 5DQC). All strong hydrogen bonding interactions are shown as dotted lines.

inhibitors. For these studies, MIN6 cells were grown in the

presence of various inhibitors, lysed and subjected to Western

blot using a monoclonal antibody vs. Tmem27 C-terminal region.

The results are shown in Figure 5.¹⁴As can be seen, the 22-kDa

C-terminal fragment of Tmem27 was formed only in small extent

in the presence of 0.4 μM of inhibitor 2a (less than 5%, lane 3).

Then, in the presence of 0.9 μM inhibitor 2a, the processing of

Tmem27 was completely abolished as shown in lane 4. We used

inhibitor 20, also known as compound J (BACE2 K_i= 6 nM;

BACE1 K_i= 18 nM) as a positive control and the results are shown

in lane 2.¹⁴

With the assumption that BACE2 processes its protein

substrate PMEL in endosomes⁵⁴these results indicated that

inhibitor 2a penetrated MIN6 cells and blocked the processing of

Tmem27 by BACE2. We now have greatly extended our

structure-activity relationships and have further investigated

determinants of inhibitors’ potency and selectivity towards BACE2

through the design, synthesis and biological evaluation of a series

of new inhibitors, namely 2f-j and 3a-o. First of all, we

synthesised a focused subset of derivatives (compounds 2f-j,

Table 1) in search of an effective P2-P3 ligand. Accordingly, we

incorporated the isophthalamide terminal nitrogen in a substituted

pyrrolidyl moiety (compounds 2f,g) and replaced the P2

benzylamine portion with an isosteric thiazolylamine moiety

(compounds 2h-j). We speculated that suitable heterocyclic

moieties and their substituents could form favorable hydrogen

bonds with backbone atoms as well as stronger van der Waals

interactions in the active site of BACE2. In particular, we selected

a 3-(R)-2-(methoxymethyl)pyrrolidinylisophtalamide and a ((4-

methylthiazol-2-yl)methyl)-isophthalamide moiety in combination

with a methyl or a longer propyl chain at the P1’ alkylamide portion.

In

compounds

2f,g

bearing

a

3-((R)-2-

(methoxymethyl)pyrrolidinylisophtalamide moiety, the insertion of

a P1’ propyl chain and a methyl group on 5-position of the

isophthalamide benzene ring led to a significant increase of

BACE1 and BACE2 activity. Accordingly, inhibitor 2g with a propyl

P1'-side chain and a 3-methyl group on the P2-isophthalamide

ligand led to improvement of both BACE1 and BACE2 inhibitory

activity. However, inhibitor 2g is more potent against BACE1 (K_i=

6.6 nM) than BACE2 (K_i= 27.9 nM). Compound 2h with a ((4-

methylthiazol-2-yl)methyl)isophthalamide moiety as the P3-ligand

showed a BACE2 K_iof 97 nM and displayed 2-fold selectivity over

BACE1. Inhibitor 2i with a longer P1' alkyl chain in combination

with no substitution on the isophthalamide phenyl ring showed

improvement of BACE1 and BACE2 potency. Compound 2j with

a

methyl group on the isophthalamide group, displayed

comparable BACE1 and BACE2 activity, however, no

Figure 4. A stereoview of an energy-minimized model of 2a (carbon chain, green) bound within the BACE2 active site. All strong

hydrogen bonding interactions are shown as dotted lines. The docking was performed with AutoDock Vina (see

SupplementaryInformation for details).

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)

M

Table 1. Structure and activity on BACE1 and BACE2 enzymes

for title inhibitors 2a-j.



9

.

0

(

K_i(nM)^a

K_i(nM)^b

a

Inhibitor

2

BACE1

BACE2

d

p

Additions

C

1

2

3

4

5

Full lengthth

Full leng

Tmem27

45.6

14.6

80.5

C-f_Cra_-fg_ram_gme_en_nt_t

of Tmem27

1812

Figure 5. Inhibition of BACE2 processing of Tmem27 by CpdJ and

compound 2a (at two different concentrations) in MIN6 cells. Lanes 1

and 5 represent vehicle (DMSO) controls with and without DAPT,

respectively.

731

182.8

0.031

improvement of selectivity over BACE1 was observed.

Based on our structural insights, we then examined a small

series on inhibitors combining the ((R)-1-1phenylethyl)

isophthalamide as the P2-P3 ligand and a P1' ligand containing a

5244

reduced peptide character. Thus, we incorporated

a

stereochemically defined aminoalcohol moiety as a convenient

handle to insert an alkyl chain of variable length similar to

inhibitors 2d and 2e coupled with a substituted phenyl ring in

place of isobutylamide. Beyond improving hydrophobic

interactions, such an aryl moiety could also serve to mimic the P2’

isobutylamide interaction with a suitably positioned polar group.

Accordingly, we synthesized compounds 3a-h (Table 2). In

general, this set of compounds showed decreased affinity

towards BACE2 compared to their terminal isobutylamide

counterparts such as inhibitors 2d and 2e. Inhibitor 3a, containing

a 3-sulfonamide group on the isophthalic moiety displayed higher

affinity towards the BACE1 enzyme (K_i= 205 nM for BACE1 and

K_i= 731 nM for BACE2). The amide N-methylation in inhibitor 3b

led to higher affinity for BACE2 with slight improvement of potency

and selectivity toward BACE2 over BACE1 (K_i= 4852 nM for

BACE1 and K_i= 479 nM for BACE2).

563.5

0.038

OH

Me

OH

H

N

H

N

3022

6.6

1366

27.9

O

MeO

O

N

Ph

H

2f

The removal of the sulfonamide functionality on the

isophthalamide phenyl ring (compounds 3c and 3d), did not

improve potency but did increase BACE2 selectivity (54-fold for

compound 3c and 34-fold for compound 3d). In agreement with

the previous series of compounds, a longer P1’ alkyl chain had a

beneficial effect for BACE2 inhibitory activity. Compound 3f,

bearing a methyl substituent on the isophthalic phenyl ring and a

176.2

33.1

96.9

51.1

terminal

(1R,2S)-1-amino-1-(3-methoxyphenyl)pentan-2-ol

displayed BACE2 inhibitory potency with a K_ivalue of 141 nM.

Interestingly, compound 3e without a methyl group on the

isophthalamide group, showed a more marked selectivity on

BACE2 over BACE1 (159-fold for 3e and 62-fold for 3f). The

removal of the 3-methoxy substituent from the P1'-ligand led to

compound 3g. However, this compound displayed a significant

drop in BACE2 inhibitory activity. Similarly, removal of the 3-

methoxy group from the P1'-ligand of inhibitor 3f resulted in

34.2

25.3

^aK_ivalues for BACE1 enzyme have been determined as described in ref.

36; ^bK_ivalues for BACE2 enzyme have been determined as described in

ref. 36.

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displayed slight improvement of BACE2 activity (K_ivalue of 35.7

nM). However, compound 3k showed reduction of BACE2

selectivity compared to inhibitor 3i (50-fold for 3i versus 37-fold

for 3k). Interestingly, incorporation of a 3-methyl group on the P2-

isophthalamide group of inhibitor 3k resulted in a very potent and

selective inhibitor 3l. Inhibitor 3l exhibited a BACE2 K_ivalue of 1.6

nM. Furthermore, inhibitor 3l showed over 500-fold selectivity for

Table 2. Structure and activity on BACE1 and BACE2 enzymes for

title inhibitors 3a-h.

K_i(nM)^a

K_i(nM)^b

Inhibitor

BACE1

BACE2

205.3

4852

730.8

BACE2. The combination of

yl)methyl)isophthalamide derivative

a

P2 ((4-methyloxazol-2-

and P1’ 3-

a

methoxybenzylamino moiety provided compound 3m. This

inhibitor showed a dramatic loss of BACE2 potency but very

potent BACE1 activity (3m, K_i= 69 nM for BACE1 and K_i= 7.3 μM

for BACE2) with selectivity over 100-fold against BACE2.

479

Table 3. Structure and activity on BACE1 and BACE2 enzymes for

title inhibitors 3i-o.

Me

N

OH

Me

OH

H

N

H

N

51331

16229

950

480

Me

O

Ph

K_i(nM)^a

BACE1

K_i(nM)^b

BACE2

Inhibitor

3c

OMe

2085

39.45

837

1347

815.1

68.8

14.02

35.71

1.6

38633

8805

243

141

42485

2552

CH₃

Me

OH

Me

N

OH

7332

H

N

18130

905

Me

O

Ph

3h

^aK_ivalues for BACE1 enzyme have been determined as described in ref. 36;

^bK_ivalues for BACE2 enzyme have been determined as described in ref. 36.

25.36

767

1921

801

compound 3h which also showed reduction of BACE2 K_ivalue of

905 nM over 3f (K_ivalue of 141 nM). These results indicated that

the 3-methoxy substituent on the phenyl ring moiety may be

involved in significant interactions in the active site.

Bearing in mind the data on prime and non-prime site

preferences, we investigated a series of compounds (3i-o, Table

3) where we removed the 3-sulfonamide functionality from the P2

isophthalamide ligand and N-methylated the amide NH of our

^aK_ivalues for BACE1 enzyme have been determined as described in ref. 36;

^bK_ivalues for BACE2 enzyme have been determined as described in ref. 36.

initial lead inhibitor 1. Compound 3i with

a

3-

methoxylbenzylamine moiety as the P1'-ligand improved BACE2

potency with a K_ivalue of 39.4 nM. This compound showed over

52-fold selectivity for BACE2. Incorporation of a 3-methyl group

on the isophthalamide group resulted in inhibitor 3j which showed

nearly 3-fold improvement of BACE2 K_i. Also, it showed slight

improvement of BACE2 selectivity (nearly 60-fold). Replacement

of the 3-methoxy group on the P1'-ligand with a lipophilic 3-

trifluoromethyl group resulted in inhibitor 3k. This inhibitor (3k)

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Figure 6. (A) An energy-minimized model of inhibitor 2d (carbon chain, green) bound in the BACE2 active site. All strong

hydrogen bonding interactions are shown as dotted lines. (B) Overlay of inhibitor 2d model with the X-ray crystal structure of

hydroxyethylamine transition state inhibitor (carbon chain, orange) in BACE2 active site (PDB : 2EWY). The docking was

performed with AutoDock Vina (see SupplementaryInformation for details).

Compound 3n, with a methyl substituent on the isophthalamide

ring, further improved BACE1 activity with a K_iof 25 nM and a

selectivity >75-fold against BACE2. Based on the efficacy of the

α-methyl group on the benzylisophthalamide moiety, we sought

to explore the outcome of an α-methyl functionality on the

oxazole-based inhibitors. Accordingly, compound 3o was

synthesized as a mixture of diastereomers (1:1) on the methyl

bearing center on the oxazolylmethyl group. This compound

exhibited comparable BACE1 and BACE2 activity with no

appreciable selectivity.

To obtain insight into the molecular binding properties

responsible for potency and selectivity of inhibitors 2d and 3l, we

created energy-minimized active models for these inhibitors as

shown in Figure 6A and 7A. The resulting models were selected

based upon the X-ray structure of the protein-ligand complex of

BACE2 (Figure 6B and 7B). Figure 6B depicts an overlay of the

inhibitor 2d model and the X-ray crystal structure of a known

hydroxyethylamine transition state inhibitor in the BACE2 active

site. Inhibitor 2d shows a similar binding orientation as the

hydroxyethylamine transition state inhibitor in the crystal structure

(also applied to 3l in Figure 7B). The detailed docking procedures

are shown in the supporting information. As can be seen in Figure

6A, inhibitor 2d makes extensive contacts in the S2′ and S3′

subsites. The P1′- NH is within proximity to form hydrogen bonds

with the Gly50 backbone NH. The P2′-carbonyl as well as P2′-

NH are also within proximity to form hydrogen bonds with Thr88

backbone NH and side chain hydroxyl groups, respectively.

Furthermore, the P3′-hydroxyl group is oriented toward Tyr211

hydroxyl group to form a hydrogen bond. The (S)-propyl group

occupies a hydrophobic pocket surrounding Lys86, Ile239, and

Tyr211. The P2 carboxamide carbonyl functionality is within

proximity to form a hydrogen bond with Gln89 and the amide NH

is also within hydrogen bonding distance with Thr244 side chain

hydroxyl group. The P3-phenylmethyl group forms extensive

hydrophobic interactions with Trp131, Thr245, and Phe125.

Overall, the number of polar interactions of P2 and P3-ligands are

significantly less compared to inhibitor 2a interactions with the

BACE1 active site. The major difference between inhibitors 2d

and 3l is that the inhibitor 3l contains a 3-trifluoromethylbenzyl

group as the P2'-ligand in place of (2S,3S)-2-amino-3-hydroxy-N-

isobutylhexanamide group as the P2-P3 ligand for 2d. The P2'-

ligand appears to make extensive hydrophobic interactions in the

Figure 7. (A) An energy-minimized model of inhibitor 3l (carbon chain, turquoise) bound in the BACE2 active site. All strong

hydrogen bonding interactions are shown as dotted lines. (B) Overlay of inhibitor 3l model with the X-ray crystal structure of

hydroxyethylamine transition state inhibitor (carbon chain, orange) in BACE2 active site (PDB : 2EWY). The docking was

performed with AutoDock Vina (see SupplementaryInformation for details).

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S2’-subsite. The interactions of P2 and P3-ligands are similar to

inhibitor 2d. The combination of active site interactions of

inhibitors 2d and 3l with BACE2 may be responsible for their high

BACE2 potency and selectivity.

analysis was performed on an Agilent 1260 Infinity instrument. All

test inhibitors showed purity >95% by HPLC analysis.

N-((2S,3R)-3-Hydroxy-4-(((2S,3S)-3-hydroxy-1-

(isobutylamino)-1-oxobutan-2-yl)amino)-1-phenylbutan-2-

yl)-3-((R)-2-(methoxymethyl)pyrrolidine-1-

carbonyl)benzamide (2f). Carboxylic acid 5a (16.5 mg, 0.063

mmol) was dissolved in dry CH₂Cl₂(3.0 mL), then EDCI (22 mg,

0.114 mmol), HOBt (15.4 mg, 0.114 mmol) and N,N-DIPEA (20

μL, 0.114 mmol) were sequentially added. Isosteric amine 10a

(0.057 mmol) dissolved in dry CH₂Cl₂(2 mL) was then added and

the reaction mixture was stirred at 25 °C for 14 h. Saturated

aqueous sodium bicarbonate was added and the reaction mixture

was extracted three times with CH₂Cl₂. The organic layers were

dried over anhydrous sodium sulfate, filtered and concentrated

under reduced pressure. The residue was purified by silica gel

flash chromatography (8% MeOH in CH₂Cl₂) affording the title

compound as a white amorphous solid (45% yield). δ ¹H NMR

(400 MHz, CDCl₃) δ 8.08 – 7.04 (m, 11H), 4.41 (br, 1H), 4.15 (br,

1H), 3.83 – 3.50 (m, 2H), 3.56 – 3.21 (m, 4H), 3.21 – 2.63 (m, 6H),

2.26 – 1.63 (m, J = 79.7 Hz, 6H), 1.65 – 1.02 (m, 10H), 0.86 (s,

6H); LRMS-ESI (m/z): 583.3 [M+H]⁺; HRMS-APCI (m/z): [M+H]⁺

calcd for C₃₂H₄₇N₄O₆, 583.3490; found 583.3496.

Conclusions

Selective BACE2 inhibitors represent a potentially new treatment

for type 2 diabetes. We present here the design, synthesis, and

evaluation of a series of potent and highly selective BACE2

inhibitors. We have utilized the X-ray structural studies with

BACE1 along with computational docking approaches with

BACE2 to optimize the potency and selectivity. Furthermore, we

have carried out an extensive SAR analysis to investigate

determinants of inhibitors’ potency and selectivity towards BACE2

enzyme using inhibitors 1 and 2a as the starting points. We have

shown that inhibitor 2a blocked the processing of Tmem27 in the

pancreatic beta-cell line MIN6. The X-ray structure of 2a-BACE1

complex was determined and then used to computationally

generate a working model of 2a bound in the BACE2 active site.

Based upon our SAR studies and structural requirements for

BACE2 selectivity, we have designed selective inhibitors 2d and

3l by removing the P2-sulfonamide, while incorporating an N-

methyl amide on the non-prime side. For the most selective

inhibitor 2d, we incorporated a propyl chain on the prime side in

inhibitor 2a. On the other hand, for inhibitor 3l, we incorporated a

highly lipophilic 3-trifluoromethylphenyl ring. Inhibitor 3l has

shown excellent BACE2 potency and high selectivity over BACE1.

Further studies including the design of more selective BACE2

inhibitors are in progress.

N-((2S,3R)-3-Hydroxy-4-(((2S,3S)-3-hydroxy-1-

(isobutylamino)-1-oxohexan-2-yl)amino)-1-phenylbutan-2-

yl)-3-((R)-2-(methoxymethyl)pyrrolidine-1-carbonyl)-5-

methylbenzamide (2g). Title compound was obtained from acid

5b and isosteric amine 10b following the procedure described for

compound 2f. ¹H NMR (400 MHz, CDCl₃) δ 7.84 (s, 1H), 7.49 (s,

1H), 7.40 (s, 2H), 7.37 – 7.10 (m, 7H), 4.48 – 4.33 (m, 2H), 3.90

– 3.78 (m, 1H), 3.78 – 3.70 (m, 1H), 3.69 – 3.53 (m, 3H), 3.36 (s,

3H), 3.31 – 3.21 (m, 1H), 3.17 (s, 1H), 3.13 – 2.91 (m, 6H), 2.89

– 2.73 (m, 2H), 2.30 (s, 2H), 2.12 – 1.84 (m, 4H), 1.82 – 1.64 (m,

2H), 1.57 – 1.28 (m, 4H), 0.98 – 0.74 (m, 9H); ¹³C NMR (200 MHz,

CDCl₃) δ 172.8), 169.5, 168.0, 138.3, 138.0, 136.9, 134.9, 130.3,

129.7, 129.3, 128.5, 126.5, 123.1, 72.5, 72.0, 71.3, 67.2, 59.2,

57.0, 55.1, 50.7, 50.3, 46.6, 36.5, 35.3, 29.7, 28.5, 27.6, 25.1,

21.2, 20.2, 19.1, 14.0. LRMS-ESI (m/z): 625.3 [M+H]⁺; HRMS-

APCI (m/z): [M+H]⁺calcd for C₃₅H₅₃N₄O₆, 625.3960; found

625.3972.

Experimental Section

General

Reactions were carried out under an argon atmosphere in either

flame- or oven-dried (120 °C) glassware. All reagents and

chemicals were purchased from commercial suppliers and used

without further purification unless otherwise noted. Anhydrous

solvents were obtained as follows: dichloromethane from calcium

hydride, diethyl ether and tetrahydrofuran from sodium

benzophenone, methanol and ethanol from activated magnesium

under argon. All purification procedures were carried out with

reagent-grade solvents (purchased form VWR) in air. TLC

analysis was conducted using glass-backed thin-layer silica gel

chromatography plates (60 a, 250 mm thickness, F254 indicator).

Column chromatography was performed using 230–400 mesh, 60

a pore diameter silica gel. ¹H and ¹³C NMR spectra were recorded

at room temperature on a Bruker AV800, DRX-500 and ARX-400

or a Varian INOVA300. Chemical shifts ( values) are reported in

parts per million and are referenced to the deuterated residual

solvent peak. NMR data are reported as:  value (chemical shift,

J-value (Hz), integration, where s = singlet, d = doublet, t = triplet,

q = quartet, brs = broad singlet). Low resolution mass analyses

were performed on an Agilent 1290 Infinity II spectrometer. High

Resolution mass analyses were performed at the Purdue

University Campus-wide Mass Spectrometry Center. HPLC

N¹-((2S,3R)-3-Hydroxy-4-(((2S,3S)-3-hydroxy-1-

(isobutylamino)-1-oxobutan-2-yl)amino)-1-phenylbutan-2-

yl)-N³,5-dimethyl-N³-((4-methylthiazol-2-

yl)methyl)isophthalamide (2h). Title compound was obtained

from acid 6b and isosteric amine 10a following the procedure

described for compound 2f. ¹H NMR (400 MHz, CDCl₃) δ 7.99 –

7.69 (m, 1H), 7.69 – 7.45 (m, 2H), 7.46 – 7.01 (m, 7H), 6.96 –

6.79 (m, 1H), 5.06 – 4.70 (m, 2H), 4.50 – 4.34 (m, 1H), 4.26 –

3.96 (m, 3H), 3.97 – 3.74 (m, 2H), 3.67 (s, 1H), 3.48 – 3.24 (m,

1H), 3.24 – 2.98 (m, 6H), 2.91 (s, 2H), 2.55 – 2.42 (m, 3H), 2.34

(s, 3H), 1.88 – 1.73 (m, 1H), 1.24 (d, J = 6.0 Hz, 3H), 1.02 – 0.83

(m, 6H); ¹³C NMR (200 MHz, CDCl₃) δ 172.6, 171.0, 167.6, 165.0,

138.9, 137.6, 134.7, 130.4, 129.7, 129.2, 128.7, 126.7, 122.8,

114.8, 71.5, 68.3, 67.7, 54.7, 50.5, 48.8, 46.6, 37.7, 36.5, 29.7,

28.5, 21.2, 20.2, 19.1, 17.0. LRMS-ESI (m/z): 624.3 [M+H]⁺;

HRMS-APCI (m/z): [M+H]⁺calcd for C₃₃H₄₆N₅O₅S, 624.3214;

found 624.3220.

N¹-((2S,3R)-3-Hydroxy-4-(((2S,3S)-3-hydroxy-1-

(isobutylamino)-1-oxohexan-2-yl)amino)-1-phenylbutan-2-

yl)-N³-methyl-N³-((4-methylthiazol-2-

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yl)methyl)isophthalamide (2i). Title compound was obtained

from acid 6a and isosteric amine 10b following the procedure

described for compound 2f. ¹H NMR (400 MHz, CDCl₃) δ 7.80 –

7.68 (m, 2H), 7.56 (d, J = 6.8 Hz, 1H), 7.41 (t, J = 7.7 Hz, 1H),

7.32 – 7.01 (m, 7H), 6.89 (s, 1H), 4.89 (br, 2H), 4.54 – 4.36 (m,

1H), 3.86 (dd, J = 11.4, 5.1 Hz, 1H), 3.76 (d, J = 4.1 Hz, 1H), 3.18

– 2.93 (m, 8H), 2.91 – 2.73 (m, 2H), 2.46 (s, 3H), 1.77 (dt, J =

13.4, 6.7 Hz, 1H), 1.60 – 1.19 (m, 6H), 1.01 – 0.83 (m, 9H); ¹³C

NMR (200 MHz, CDCl₃) δ 172.6, 170.8, 167.2, 164.9, 152.5,

137.7, 135.4, 134.9, 129.9, 129.6, 129.5, 129.2, 128.6, 127.8,

126.7, 125.9, 114.8, 113.9, 109.8, 72.1, 71.1, 67.0, 54.8, 50.3,

48.9, 46.6, 37.8, 36.6, 35.5, 29.7, 28.4, 20.2, 19.1, 17.0, 14.0.

LRMS-ESI (m/z): 638.3 [M+H]⁺; HRMS-APCI (m/z): [M+H]⁺calcd

for C₃₄H₄₈N₅O₅S, 638.3371; found 638.3380.

compound was obtained from acid 4a and the free amine of Boc

compound 17a following the procedure described for compound

2f. ¹H NMR (500 MHz, CDCl₃) δ 7.84 (d, J = 7.5 Hz, 2H), 7.54 (br,

1H), 7.51 – 7.15 (m, 13H), 6.95 (d, J = 8.5 Hz, 2H), 6.92 – 6.83

(m, 1H), 6.21 (s, 0.5H), 5.03 (s, 0.5H), 4.52 (s, 1H), 4.19 (s, 1H),

3.94 – 3.81 (m, 3H), 3.81 – 3.68 (m, 1H), 3.67 – 3.57 (m, 1H),

3.11 (s, 2H), 2.99 (dd, J = 14.1, 6.3 Hz, 2H), 2.95 – 2.70 (m, 4H),

2.63 (s, 2H), 1.77 – 1.52 (m, 3H), 1.14 – 1.05 (m, 3H); ¹³C NMR

(125 MHz, CDCl₃) δ 171.3, 167.7, 160.1, 140.9, 138.2, 137.1,

129.8, 130.0, 128.8, 128.1, 127.8, 127.0, 125.9, 121.1, 114.6,

113.2, 71.0, 70.4, 68.9, 55.6, 55.4, 49.6, 37.4, 20.1, 15.8. LRMS-

ESI (m/z): 610.0 [M+H]⁺, 632.2 [M+H]⁺; HRMS-ESI (m/z): [M+H]⁺

calcd for C₃₇H₄₄N₃O₅, 610.3276; found 610.3270.

N¹-((2S,3R)-3-Hydroxy-4-(((1R,2S)-2-hydroxy-1-(3-

N¹-((2S,3R)-3-Hydroxy-4-(((2S,3S)-3-hydroxy-1-

(isobutylamino)-1-oxohexan-2-yl)amino)-1-phenylbutan-2-

yl)-N³,5-dimethyl-N³-((4-methylthiazol-2-

methoxyphenyl)propyl)amino)-1-phenylbutan-2-yl)-N³,5-

dimethyl-N³-((R)-1-phenylethyl)isophthalamide (3d). Title

compound was obtained from acid 4b and the free amine of Boc

compound 17a following the procedure described for compound

2f. ¹H NMR (500 MHz, CDCl₃) δ 7.70 – 7.57 (m, 2H), 7.54 – 7.17

(m, 13H), 7.08 – 6.84 (m, 3H), 6.21 (s, 0.5H), 5.03 (s, 0.5H), 4.51

(s, 1H), 4.19 (s, 1H), 3.85 (s, 3H), 3.80 – 3.68 (m, 1H), 3.62 (s,

1H), 3.10 (s, 1H), 3.07 – 2.86 (m, 3H), 2.86 – 2.70 (m, 3H), 2.62

(s, 2H), 2.44 (s, 3H), 1.75 – 1.58 (m, 3H), 1.11 (d, J = 6.3 Hz, 3H);

¹³C NMR (125 MHz, CDCl₃) δ 168.0, 160.1, 140.7, 138.1, 137.2,

135.4, 130.2, 129.6, 129.4, 129.0, 128.1, 127.8, 127.1, 122.9,

121.2, 114.6, 113.3, 71.1, 70.4, 68.9, 55.6, 55.3, 49.5, 37.3, 30.2,

21.8, 20.2. LRMS-ESI (m/z): 624.3 [M+H]⁺; HRMS-APCI (m/z):

[M+H]⁺calcd for C₃₈H₄₆N₃O₅, 624.3432; found 624.3437.

yl)methyl)isophthalamide (2j). Title compound was obtained

from acid 6b and isosteric amine 10b following the procedure

described for compound 2f. ¹H NMR (400 MHz, CDCl₃) δ 7.64 –

7.46 (m, 2H), 7.44 – 7.17 (m, 6H), 7.10 (s, 2H), 6.89 (s, 1H), 4.91

(s, 2H), 4.43 (s, 1H), 3.87 (s, 1H), 3.74 (s, 1H), 3.33 – 2.94 (m,

9H), 2.84 (s, 2H), 2.46 (s, 3H), 2.37 (s, 3H), 1.86 – 1.70 (m, 1H),

1.55 – 1.18 (m, 6H), 1.03 – 0.81 (m, J = 6.5 Hz, 9H); ¹³C NMR

(200 MHz, CDCl₃) δ 172.7, 171.0, 167.6, 165.0, 152.5, 138.9,

137.7, 135.4, 134.8, 130.1, 129.6, 129.5, 129.3, 128.6, 126.7,

122.8, 114.8, 113.9, 72.1, 71.2, 67.0, 54.7, 48.9, 46.6, 37.8, 36.6,

35.5, 29.7, 28.5, 21.2, 20.2, 19.1, 17.0, 14.1, 13.9. LRMS-ESI

(m/z): 652.3 [M+H]⁺; HRMS-APCI (m/z): [M+H]⁺calcd for

C₃₅H₅₀N₅O₅S, 652.3527; found 652.3531.

N¹-((2S,3R)-3-Hydroxy-4-(((1R,2S)-2-hydroxy-1-(3-

methoxyphenyl)pentyl)amino)-1-phenylbutan-2-yl)-N³-

N¹-((2S,3R)-3-Hydroxy-4-(((1R,2S)-2-hydroxy-1-(3-

methoxyphenyl)propyl)amino)-1-phenylbutan-2-yl)-5-(N-

methylmethylsulfonamido)-N³-((R)-1-

methyl-N³-((R)-1-phenylethyl)isophthalamide

(3e).

Title

compound was obtained from acid 4a and the free amine of Boc

compound 17b following the procedure described for compound

2f. ¹H NMR (800 MHz, CDCl₃) δ 7.96 – 7.73 (m, 2H), 7.51 (d, J =

7.5 Hz, 1H), 7.46 – 7.37 (m, 4H), 7.34 (dd, J = 15.2, 7.5 Hz, 1H),

7.28 – 7.22 (m, 5H), 7.22 – 7.14 (m, 2H), 6.92 (d, J = 7.9 Hz, 2H),

6.85 – 6.82 (m, 1H), 6.16 (s, 0.5H), 5.01 (s, 0.5H), 4.48 (s, 1H),

3.97 (s, 1H), 3.82 (s, 3H), 3.73 (dd, J = 12.6, 3.2 Hz, 1H), 3.61 (d,

J = 1.9 Hz, 1H), 3.09 (s, 1H), 2.97 (dd, J = 14.1, 6.3 Hz, 1H), 2.92

– 2.81 (m, 1H), 2.84 – 2.69 (m, 2H), 2.59 (s, 1H), 1.70 – 1.56 (m,

3H), 1.40 – 1.25 (m, 4H), 1.19 (td, J = 9.1, 4.7 Hz, 1H), 0.89 –

0.80 (m, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 167.5, 159.9, 140.8,

138.0, 136.9, 129.8, 129.6, 129.8, 128.7, 127.9, 127.7, 126.9,

125.8, 121.0, 114.5, 113.0, 70.9, 70.3, 68.8, 55.5, 55.30, 53.8,

49.5, 37.2, 32.2, 20.0, 15.7. LRMS-ESI (m/z): 638.3 [M+H]⁺;

HRMS-APCI (m/z): [M+H]⁺calcd for C₃₉H₄₈N₃O₅, 638.3589; found

638.3596.

phenylethyl)isophthalamide (3a). Title compound was obtained

from acid 4d and the free amine of Boc compound 17a following

1

the procedure described for compound 2f. H NMR (300 MHz,

CDCl₃) δ 8.62 (d, J = 8.0 Hz, 1H), 8.15 (s, 1H), 7.94 – 7.83 (m,

2H), 7.42 – 7.12 (m, 13H), 7.04 (br, 1H), 6.87 – 6.79 (m, 3H), 5.37

– 5.23 (m, 1H), 4.56 – 4.40 (m, 1H), 4.15 (dd, J = 6.4, 3.7 Hz, 1H),

3.84 – 3.77 (m, 3H), 3.75 – 3.65 (m, 1H), 3.51 (d, J = 3.5 Hz, 1H),

3.33 – 3.27 (m, 3H), 3.05 (dd, J = 13.9, 7.5 Hz, 1H), 2.88 – 2.75

(m, 5H), 2.75 – 2.64 (m, 1H), 1.58 (d, J = 6.9 Hz, 3H), 0.95 (d, J

= 6.4 Hz, 5H).

N¹-((2S,3R)-3-Hydroxy-4-(((1R,2S)-2-hydroxy-1-(3-

methoxyphenyl)propyl)amino)-1-phenylbutan-2-yl)-N³-

methyl-5-(N-methylmethylsulfonamido)-N³-((R)-1-

phenylethyl)isophthalamide (3b). Title compound was obtained

from acid 4c and the free amine of Boc compound 17a following

N¹-((2S,3R)-3-Hydroxy-4-(((1R,2S)-2-hydroxy-1-(3-

1

the procedure described for compound 2f. H NMR (300 MHz,

methoxyphenyl)pentyl)amino)-1-phenylbutan-2-yl)-N³,5-

dimethyl-N³-((R)-1-phenylethyl)isophthalamide (3f). Title

compound was obtained from acid 4b and the free amine of Boc

compound 17b following the procedure described for compound

2f. ¹H NMR (300 MHz, CDCl₃) δ 7.82 (s, 1H), 7.56 (d, J = 8.8 Hz,

2H), 7.50 – 7.07 (m, 12H), 6.90 (d, J = 6.6 Hz, 2H), 6.81 (d, J =

8.1 Hz, 1H), 6.13 (s, 0.5H), 4.98 (s, 0.5H), 4.44 (s, 1H), 3.95 (s,

1H), 3.77 (s, 3H), 3.75 – 3.62 (m, 1H), 3.58 (s, 1H), 3.17 – 2.90

(m, 3H), 2.92 – 2.48 (m, 6H), 2.36 (s, 3H), 1.70 – 1.42 (m, 4H),

1.44 – 1.09 (m, 4H), 0.83 (t, J = 6.9 Hz, 3H); ¹³C NMR (125 MHz,

CDCl₃) δ 168.1, 160.2, 140.8, 138.5, 137.9, 137.3, 135.5, 130.7,

130.0, 129.1, 129.3, 128.7, 128.4, 128.1, 127.98, 127.6, 127.4,

CDCl₃) δ 7.93 – 7.72 (m, 2H), 7.59 (s, 1H), 7.48 – 7.03 (m, 12H),

6.99 – 6.72 (m, 3H), 6.11 (s, 0.5H), 5.01 (s, 0.5H), 4.49 (s, 1H),

4.20 (s, 1H), 3.79 (s, 3H), 3.77 – 3.63 (m, 2H), 3.59 (s, 1H), 3.40

– 3.20 (m, 3H), 3.17 – 2.97 (m, 2H), 2.97 – 2.69 (m, 7H), 2.70 –

2.53 (m, 2H), 1.61 (d, J = 6.8 Hz, 3H), 1.02 (d, J = 5.9 Hz, 3H).

LRMS-ESI (m/z): 717.8 [M+H]⁺; HRMS-ESI (m/z): [M+H]⁺calcd

for C₃₉H₄₉N₄O₇S, 717.3317; found 717.3310.

N¹-((2S,3R)-3-Hydroxy-4-(((1R,2S)-2-hydroxy-1-(3-

methoxyphenyl)propyl)amino)-1-phenylbutan-2-yl)-N³-

methyl-N³-((R)-1-phenylethyl)isophthalamide

(3c).

Title

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126.8, 123.0, 121.7, 121.0, 115.1, 114.3, 113.7, 113.0, 71.2, 70.4,

69.0, 55.7, 55.4, 49.6, 37.4, 32.4, 30.2, 21.8, 20.2, 18.1, 15.9.

LRMS-ESI (m/z): 652.4 [M+H]⁺; HRMS-APCI (m/z): [M+H]⁺calcd

for C₄₀H₅₀N₃O₅, 652.3745; found 652.3749.

1.72 – 1.47 (m, 3H); ¹³C NMR (200 MHz, CDCl₃) δ 170.6, 167.2,

140.1, 137.6, 137.2, 131.7, 131.4, 131.3, 130.9, 129.3, 128.8,

129.0, 127.9, 127.7, 127.4, 126.7, 126.5, 126.3, 126.4, 126.42,

125.44, 124.2, 123.4, 113.9, 70.7, 54.0, 53.3, 50.7, 36.4, 29.7,

22.70. LRMS-ESI (m/z): 604.3 [M+H]⁺; HRMS-APCI (m/z): [M+H]⁺

calcd for C₃₅H₃₇F₃N₃O₃, 604.2782; found 604.2785.

N¹-((2S,3R)-3-Hydroxy-4-(((1R,2S)-2-hydroxy-1-

phenylpentyl)amino)-1-phenylbutan-2-yl)-N³-methyl-N³-((R)-

1-phenylethyl)isophthalamide (3g). Title compound was

obtained from acid 4a and the free amine of Boc compound 17c

following the procedure described for compound 2f. ¹H NMR (300

MHz, CDCl₃) δ 8.03 (s, 1H), 7.80 (s, 2H), 7.62 – 7.10 (m, 17H),

6.13 (s, 1H), 4.98 (s, 1H), 4.56 – 4.39 (m, 1H), 3.95 (s, 1H), 3.68

(s, 1H), 3.60 (d, J = 3.3 Hz, 1H), 3.08 (dd, J = 13.8, 7.6 Hz, 2H),

2.92 (dd, J = 14.0, 6.4 Hz, 2H), 2.88 – 2.65 (m, 4H), 2.57 (s, 2H),

1.68 – 1.42 (m, 3H), 1.37 – 1.02 (m, 4H), 0.83 (t, J = 7.1 Hz, 3H);

¹³C NMR (200 MHz, CDCl₃) δ 167.4, 137.7, 136.7, 129.3, 128.8,

N¹-((2S,3R)-3-Hydroxy-1-phenyl-4-((3-

(trifluoromethyl)benzyl)amino)butan-2-yl)-N³,5-dimethyl-N³-

((R)-1-phenylethyl)isophthalamide (3l). Title compound was

obtained from acid 4b and isosteric amine 18a following the

procedure described for compound 2f. ¹H NMR (300 MHz, CDCl₃)

¹H NMR (800 MHz, CDCl₃) δ 7.62 (s, 1H), 7.56 (dd, J = 21.7, 7.5

Hz, 2H), 7.52 – 7.38 (m, 5H), 7.39 – 7.30 (m, 3H), 7.27 – 7.12 (m,

5H), 6.85 – 6.69 (m, 1H), 6.16 (br, 1H), 4.94 (br, 1H), 4.43 – 4.30

(m, 1H), 3.92 (dd, J = 37.4, 13.4 Hz, 2H), 3.79 – 3.64 (m, 1H),

128.4, 127.5, 126.6, 126.5, 73.5, 70.5, 67.5, 55.1, 49.0, 36.9, 36.3, 3.10 (dd, J = 14.2, 5.0 Hz, 2H), 2.99 (s, 1H), 2.91 – 2.69 (m, 4H),

29.7, 19.2. LRMS-ESI (m/z): 608.3 [M+H]⁺; HRMS-ESI (m/z):

2.56 (s, 2H), 2.36 (s, 3H), 1.68 – 1.49 (m, 3H); ¹³C NMR (200 MHz,

CDCl₃) δ 170.8, 167.5, 139.9, 137.6, 131.8, 131.2, 130.2, 130.4,

130.2, 130.0, 130.0, 129.3. 127.5, 127.2, 126.9, 126.6, 125.2,

124.8, 124.4, 123.4, 70.8, 53.8, 53.2, 50.6, 36.4, 29.7, 21.2.

LRMS-ESI (m/z): 618.3 [M+H]⁺; HRMS-APCI (m/z): [M+H]⁺calcd

for C₃₆H₃₉F₃N₃O₃, 618.2938; found 618.2945.

[M+H]⁺calcd for C₃₈H₄₆N₃O₄, 608.3483; found 608.3493.

N¹-((2S,3R)-3-Hydroxy-4-(((1R,2S)-2-hydroxy-1-

phenylpentyl)amino)-1-phenylbutan-2-yl)-N3,5-dimethyl-N³-

((R)-1-phenylethyl)isophthalamide (3h). Title compound was

obtained from acid 4b and the free amine of Boc compound 17c

following the procedure described for compound 2f. ¹H NMR (500

MHz, CDCl₃) δ 8.49 – 8.26 (m, J = 24.0 Hz, 2H), 8.15 (d, J = 19.2

Hz, 2H), 8.01 – 7.61 (m, 15H), 6.67 (s, 1H), 5.02 (s, 1H), 4.64 (s,

2H), 4.24 (s, 2H), 3.80 – 3.60 (m, 2H), 3.58 – 3.17 (m, 2H), 3.12

(s, 2H), 2.91 (s, 2H), 2.14 (s, 3H), 2.01 (s, 2H), 1.88 – 1.57 (m,

5H), 1.42 – 1.28 (m, 3H); ¹³C NMR (200 MHz, CDCl₃) δ 167.4,

137.6, 136.8, 130.8, 129.6, 129.4, 128.5, 127.8, 127.5, 126.6,

70.6, 67.4, 54.9, 48.8, 36.7, 36.3, 29.7, 22.7, 21.3, 19.1, 14.0.

LRMS-ESI (m/z): 622.3 [M+H]⁺; HRMS-ESI (m/z): [M+H]⁺calcd

for C₃₉H₄₇N₃O₄, 622.3640; found 622.3635.

N¹-((2S,3R)-3-Hydroxy-4-((3-methoxybenzyl)amino)-1-

phenylbutan-2-yl)-N³-methyl-N³-((4-methyloxazol-2-

yl)methyl)isophthalamide (3m). Title compound was obtained

from acid 7a and isosteric amine 18b following the procedure

described for compound 2f. ¹H NMR (800 MHz, CDCl₃) δ 7.87 (d,

J = 47.0 Hz, 1H), 7.80 – 7.62 (m, 2H), 7.63 – 7.46 (m, 1H), 7.36

(dd, J = 20.3, 12.6 Hz, 5H), 7.24 – 7.15 (m, 1H), 7.16 – 7.10 (m,

1H), 7.01 (s, 1H), 6.94 (d, J = 7.3 Hz, 1H), 6.82 (dd, J = 8.3, 2.2

Hz, 1H), 4.85 – 4.77 (m, 1H), 4.41 (s, 1H), 4.35 (d, J = 5.3 Hz, 1H),

4.13 – 4.04 (m, 2H), 4.01 – 3.94 (m, 1H), 3.72 (s, 3H), 3.15 – 3.03

(m, 3H), 2.98 – 2.86 (m, 3H), 2.22 – 2.10 (m, 3H); ¹³C NMR (200

MHz, CDCl₃) δ 167.4, 159.9, 159.3, 137.8, 136.9, 136.2, 135.8,

135.5, 135.2, 134.2, 130.0, 129.4, 128.6, 128.5, 127.8, 126.5,

126.1, 124.5, 121.7, 114.8, 114.7, 114.6, 113.9, 70.0, 55.3, 54.1,

52.3, 50.4, 35.9, 29.7. LRMS-ESI (m/z): 557.2 [M+H]⁺; HRMS-

APCI (m/z): [M+H]⁺calcd for C₃₂H₃₇N₄O₅, 557.2759; found

557.2755.

N¹-((2S,3R)-3-Hydroxy-4-((3-methoxybenzyl)amino)-1-

phenylbutan-2-yl)-N³-methyl-N³-((R)-1-

phenylethyl)isophthalamide (3i). Title compound was obtained

from acid 4a and isosteric amine 18b following the procedure

described for compound 2f. ¹H NMR (400 MHz, CDCl₃) δ 7.75 (s,

1H), 7.67 (d, J = 7.5 Hz, 1H), 7.52 (d, J = 7.4 Hz, 1H), 7.47 – 7.12

(m, 12H), 7.05 (br, 1H), 6.99 – 6.89 (m, 2H), 6.84 (d, J = 7.6 Hz,

1H), 4.49 – 4.34 (m, 1H), 3.96 – 3.74 (m, 6H), 3.72 – 3.45 (m, 2H),

3.13 – 2.97 (m, 2H), 2.93 – 2.77 (m, 2H), 2.78 – 2.57 (m, 2H),

1.62 (d, J = 6.8 Hz, 3H); ¹³C NMR (200 MHz, CDCl₃) δ 170.7,

167.2, 159.9, 137.8, 129.7, 129.3, 128.8, 128.7, 127.7, 127.5,

N¹-((2S,3R)-3-Hydroxy-4-((3-methoxybenzyl)amino)-1-

phenylbutan-2-yl)-N³,5-dimethyl-N³-((4-methyloxazol-2-

yl)methyl)isophthalamide (3n). Title compound was obtained

from acid 7b and isosteric amine 18b following the procedure

126.6, 120.9, 114.2, 113.3, 70.4, 55.3, 54.2, 53.4, 50.5, 36.4, 29.7. described for compound 2f. ¹H NMR (800 MHz, CDCl₃) δ 7.67 –

LRMS-ESI (m/z): 566.3 [M+H]⁺; HRMS-ESI (m/z): [M+H]⁺calcd

7.47 (m, 1H), 7.46 – 7.34 (m, 1H), 7.34 – 7.08 (m, 8H), 7.06 –

6.87 (m, 2H), 6.84 (d, J = 8.3 Hz, 1H), 4.82 (s, 1H), 4.44 (s, 1H),

4.41 – 4.32 (m, 1H), 3.96 (s, 1H), 3.88 (d, J = 12.0 Hz, 2H), 3.78

(s, 3H), 3.17 – 3.05 (m, 3H), 3.05 – 2.89 (m, 5H), 2.89 – 2.73 (m,

2H), 2.35 (s, 3H), 2.19 (s, 3H); ¹³C NMR (200 MHz, CDCl₃) δ

171.1, 167.5, 159.9, 159.3, 139.0, 137.8, 136.6, 135.6, 135.1,

134.3, 130.7, 129.8, 129.5, 129.3, 128.5, 126.6, 122.9, 121.2,

114.4, 113.9, 70.3, 55.3, 54.0, 52.9, 50.5, 36.2, 29.7, 21.2, 11.5.

LRMS-ESI (m/z): 571.3 [M+H]⁺; HRMS-APCI (m/z): [M+H]⁺calcd

for C₃₃H₃₉N₄O₅, 571.2915; found 571.2921.

for C₃₅H₄₀N₃O₄, 566.3013; found 566.3009.

N¹-((2S,3R)-3-Hydroxy-4-((3-methoxybenzyl)amino)-1-

phenylbutan-2-yl)-N3,5-dimethyl-N³-((R)-1-

phenylethyl)isophthalamide (3j). Compound was synthesized

as reported.^{36 1}H, ¹³C, LRMS and HRMS are identical to those

reported in literature.³⁶

N¹-((2S,3R)-3-Hydroxy-1-phenyl-4-((3-

(trifluoromethyl)benzyl)amino)butan-2-yl)-N³-methyl-N³-((R)-

1-phenylethyl)isophthalamide (3k). Title compound was

obtained from acid 4a and isosteric amine 18a following the

procedure described for compound 2f. ¹H NMR (400 MHz, CDCl₃)

δ 7.85 – 7.08 (m, 18H), 6.94 (br, 1H), 6.14 (br, 1H), 4.92 (s, 1H),

4.50 – 4.23 (m, 1H), 3.86 (q, J = 13.5 Hz, 2H), 3.75 – 3.57 (m, 1H),

3.17 – 2.90 (m, 3H), 2.91 – 2.65 (m, 4H), 2.66 – 2.43 (m, 1H),

N¹-((2S,3R)-3-Hydroxy-4-((3-methoxybenzyl)amino)-1-

phenylbutan-2-yl)-N³,5-dimethyl-N³-(1-(4-methyloxazol-2-

yl)ethyl)isophthalamide (3o): To a solution of carboxylic acid 24

(42 mg, 0.13 mmol) in CH₂Cl₂(2 mL) were added EDCI^.HCl (32

°

mg, 0.16 mmol) and HOBt^.H₂O (22 mg, 0.16 mmol) at 0 C and

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the mixture was stirred for 10 min. Amine 18b (50 mg, 0.16 mmol)

– 7.18 (m, 1H), 7.05 – 6.92 (m, 2H), 6.88 – 6.74 (m, 1H), 6.51 –

6.34 (m, 1H), 6.35 – 6.18 (m, 1H), 3.84 (s, 3H), 2.32 – 2.13 (m,

2H), 1.63 – 1.43 (m, 2H), 1.00 (t, J = 7.3 Hz, 3H).

in CH₂Cl₂(1 mL) and DIPEA (30 µL, 0.16 mmol) were added at 0

o

^oC and the resulting mixture was stirred at 23 C for 12 h. After

this period, the reaction was quenched with saturated aqueous

NH₄Cl solution. The mixture was extracted with CH₂Cl₂, dried over

anhydrous Na₂SO₄, filtered and concentrated under reduced

pressure. The residue was purified by column chromatography

over silica gel (3% MeOH/CH₂Cl₂) to afford inhibitor 3o (39 mg,

(E)-Pent-1-en-1-ylbenzene (12c). Title compound was obtained

following the procedure described for 12a. The residue was

purified by silica gel flash chromatography (n-hexane-EtOAc

15:1) to afford title compound as a dark yellow oil (81% yield over

2 steps). ¹H NMR (300 MHz, CDCl₃) δ 7.45 – 7.26 (m, 4H), 7.25

– 7.14 (m, 1H), 6.46 – 6.34 (m, 1H), 6.33 – 6.16 (m, 1H), 2.20 (td,

J = 7.6, 0.8 Hz, 2H), 1.61 – 1.40 (m, 2H), 1.08 – 0.92 (m, 3H).

1

48%). H NMR (800 MHz, CD₃OD): δ 7.70-7.61 (m, 1H), 7.53-

7.37 (m, 3H), 7.34 (t, J = 7.3 Hz, 1H), 7.30-7.21 (m, 4H), 7.17 (m,

1H), 7.10 (m, 1H), 7.07 (d, J = 6.5 Hz, 1H), 6.98 (d, J = 7.6 Hz,

1H), 5.95 (m, 0.6H), 4.96 (m, 0.4H), 4.28-4.19 (m, 3H), 3.96 (m,

1H), 3.81 (s, 3H), 3.38 (d, J = 12.5 Hz, 1H), 3.19 (d, J = 12.0 Hz,

1H), 3.06 (m, 1H), 2.89 (m, 1H), 2.86-2.73 (m, 3H), 2.42 (s, 3H),

2.23-2.14 (m, 3H), 1.74-1.56 (m, 3H); ¹³C NMR (200 MHz,

CD₃OD): δ 171.4, 168.8, 160.3, 139.1, 138.2, 136.3, 135.9, 135.7,

135.5, 134.2, 132.0, 130.3, 129.9, 129.1, 129.0, 128.7, 128.1,

126.2, 122.6, 121.8, 115.2, 114.9, 69.2, 54.6, 54.4, 50.8, 49.6,

48.1, 48.0, 35.8, 32.3, 19.8,14.9, 13.8, 9.8; LRMS-ESI (m/z): 585

[M+H]⁺; HRMS-ESI (m/z): [M+H]⁺calcd for C₃₄H₄₁N₄O₅, 585.3072;

found 585.3068.

(1S,2S)-1-(3-Methoxyphenyl)propane-1,2-diol

(13a).

Compound 12a (200 mg, 1.35 mmol) was dissolved in a 1:1

mixture of t-BuOH and water (20 mL). Methanesulfonamide (193

mg, 2.02 mmol) and AD-mix-α (1.9 g) were added in sequence

and the reaction mixture was stirred at 25 °C for 36 h. The reaction

was quenched with Na₂SO₃and extracted three times with EtOAc.

The organic layer was washed with brine, dried over anhydrous

sodium sulfate, filtered and concentrated under reduced pressure.

The residue was purified by silica gel flash chromatography (n-

hexane/EtOAc 1:1) providing title compound as

a white

amorphous solid (73% yield). ¹H NMR (300 MHz, CDCl₃) δ 7.32 –

7.16 (m, 1H), 6.96 – 6.75 (m, 3H), 4.40 – 4.23 (m, 1H), 3.89 –

3.73 (m, 4H), 3.11 – 2.96 (m, 2H), 2.80 (br, 1H), 1.04 (d, J = 6.3

Hz, 3H); ¹³C NMR (75 MHz, CDCl₃) δ 159.6, 142.6, 129.4, 119.1,

113.3, 112.3, 79.2, 72.0, 55.1, 18.6.

(2S,3S)-2-(((2R,3S)-3-Amino-2-hydroxy-4-

phenylbutyl)amino)-3-hydroxy-N-isobutylbutanamide (10a).

Compound was synthesized from derivative 8a and epoxide 9 as

reported. ¹H data are identical to those reported in literature.³⁶

(2S,3S)-2-(((2R,3S)-3-Amino-2-hydroxy-4-

(1S,2S)-1-(3-Methoxyphenyl)pentane-1,2-diol (13b). Title

compound was obtained following the procedure described for

13a. The residue was purified by silica gel flash chromatography

(n-hexane-EtOAc 1:1) to afford title compound as a dark yellow

oil (68% yield). ¹H NMR (300 MHz, CDCl₃) δ 7.29 – 7.12 (m, 1H),

6.91 – 6.70 (m, 3H), 4.42 – 4.18 (m, 1H), 3.78 – 3.65 (m, 4H),

3.11 – 2.96 (m, 2H), 2.87 (br, 1H), 1.71 – 1.23 (m, 4H), 0.98 –

0.75 (m, 3H).

phenylbutyl)amino)-3-hydroxy-N-isobutylhexanamide (10b).

Compound was synthesized from derivative 8b and epoxide 9 as

reported. ¹H data are identical to those reported in literature.³⁶

(E)-1-Methoxy-3-(prop-1-en-1-yl)benzene (12a). n-Butyllithium

(1.6 M solution in n-hexane, 6.5 mL, 10.43 mmol) was added

dropwise to a stirred suspension of ethyltriphenylphosphonium

bromide (3.8 g, 10.28 mmol) in dry THF (37 mL) at -78 °C under

argon atmosphere. The temperature of the reaction mixture was

allowed to rise to 0 °C for 30 min and then the solution was cooled

(1S,2S)-1-Phenylpentane-1,2-diol (13c). Title compound was

obtained following the procedure described for 13a. The residue

was purified by silica gel flash chromatography (n-hexane-EtOAc

again to -78 °C. Subsequently,

a

solution of 3-

methoxybenzaldehyde 11a (1.0 g, 7.35 mmol) in dry THF (10 mL)

was added over 30 min. The reaction mixture was stirred for an

additional 20 min at -78 °C and then for 3 h at 25 °C. Water was

added and the reaction mixture was extracted three times with

EtOAc. The organic layers were dried over anhydrous sodium

sulfate, filtered and concentrated under reduced pressure. The

residue was purified by silica gel flash chromatography (n-

1

1:1) to afford title compound as a dark yellow oil (68% yield). H

NMR (300 MHz, CDCl₃) δ 7.49 – 7.24 (m, 5H), 4.44 (d, J = 6.8 Hz,

1H), 3.70 (ddd, J = 8.3, 6.9, 3.5 Hz, 1H), 2.50 (br, 2H), 1.62 – 1.19

(m, 4H), 0.96 – 0.78 (m, 3H).

(4S,5S)-4-Methyl-5-(3-methoxyphenyl)-1,3,2-dioxathiolane-2-

oxide (14a). Diol 13a (300 mg, 1.65 mmol) was dissolved in

triethylamine (5 mL) and cooled to 0 °C in an ice bath under an

argon atmosphere. Thionyl chloride (144 L, 1.98 mmol) was

added dropwise and the reaction mixture stirred at 0 °C for 50 min

(monitored by TLC). After completion, ice-cold water (20 mL) was

added and the mixture was extracted with Et₂O. The ethereal

layer was washed with 10% HCl, saturated aqueous NaHCO₃and

brine. The organic layer was dried over anhydrous sodium sulfate,

filtered and evaporated under reduced pressure. The residue was

purified by silica gel flash chromatography (n-hexane-EtOAc 9:1)

providing the compound as a dark yellow oil (79% yield, mixture

hexane-EtOAc

9:1)

providing

1-methoxy-3-(prop-1-en-1-

yl)benzene as E/Z mixture (88% yield). The E/Z mixture of

alkenes (959 mg, 6.41 mmol) was dissolved in chloroform (40 mL).

Bis(acetonitrile)dichloropalladium (II) (30 mg) was added and the

reaction mixture was stirred at 25 °C for 72 h. Then, solvent was

evaporated, and the residue was purified by using a short silica

gel path eluted with n-hexane-EtOAc 9:1 to afford 12a as a pale

1

yellow oil (95% yield). H NMR (300 MHz, CDCl₃) δ 7.24 (t, J =

7.9 Hz, 1H), 7.01 – 6.85 (m, 2H), 6.78 (dd, J = 8.2, 2.5 Hz, 1H),

6.50 – 6.35 (m, 1H), 6.36 – 6.16 (m, 1H), 3.83 (s, 3H), 1.91 (d, J

= 6.3 Hz, 3H).

of diastereoisomers). ¹H NMR (300 MHz, CDCl₃)

δ (1:1

diastereomeric mixture) 7.33 (td, J = 7.9, 3.1 Hz, 1H), 7.09 – 6.88

(m, 3H), 5.41 (d, J = 9.1 Hz, 0.5H), 4.93 – 4.77 (m, 1H), 4.39 (dq,

J = 9.0, 6.2 Hz, 0.5H), 3.83 (s, 3H), 1.60 (t, J = 10.4 Hz, 1.5H),

1.52 – 1.43 (m, 1.5H).

(E)-1-Methoxy-3-(pent-1-en-1-yl)benzene

(12b).

Title

compound was obtained following the procedure described for

12a. The residue was purified by silica gel flash chromatography

(n-hexane-EtOAc 9:1) to afford title compound as a dark yellow

oil (69% yield over two steps). ¹H NMR (300 MHz, CDCl₃) δ 7.33

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(4S,5S)-4-Propyl-5-(3-methoxyphenyl)-1,3,2-dioxathiolane-2-

oxide (14b). Title compound was obtained following the

procedure described for 14a. The residue was purified by silica

gel flash chromatography (n-hexane-EtOAc 9:1) to afford title

compound as a brown oil (66% yield). ¹H NMR (300 MHz, CDCl₃)

δ (1:1 diastereomeric mixture) 7.32 (td, J = 7.8, 3.0 Hz, 1H), 6.96

(ddd, J = 13.3, 9.2, 5.9 Hz, 3H), 5.45 (d, J = 9.0 Hz, 0.5H), 4.88

(d, J = 9.6 Hz, 0.5H), 4.74 (dt, J = 9.6, 6.0 Hz, 0.5H), 4.31 (td, J =

8.8, 3.7 Hz, 0.5H), 3.81 (s, 3H), 2.00 – 1.32 (m, 4H), 0.92 (td, J =

7.2, 2.1 Hz, 3H).

evaporated under reduced pressure. This operation was repeated

additional two times. The residue was then taken up in CH₂Cl₂

and saturated aqueous NaHCO₃(1 mL) was added. The organic

layer was dried over anhydrous sodium sulfate, filtered and

concentrated under reduced pressure to afford desired

aminoalcohol as a white amorphous solid (86% yield, 2 steps).

(1R,2S)-1-Amino-1-(3-methoxyphenyl)pentan-2-ol (16b). Title

compound was obtained following the procedure described for

16a to afford title compound as a white amorphous solid (66%

yield,

2

steps).

tert-Butyl

((1R,2S)-2-hydroxy-1-(3-

(4S,5S)-4-Propyl-5-phenyl-1,3,2-dioxathiolane-2-oxide (14c).

Title compound was obtained following the procedure described

for 14a. The residue was purified by silica gel flash

chromatography (n-hexane-EtOAc 9:1) to afford title compound

as a brown oil (54% yield). ¹H NMR (300 MHz, CDCl₃) δ (1:1

diastereomeric mixture) 7.63 – 7.33 (m, 5H), 5.49 (d, J = 9.1 Hz,

0.5H), 4.91 (d, J = 9.6 Hz, 0.5H), 4.85 – 4.68 (m, 0.5H), 4.44 –

4.22 (m, 0.5H), 2.01 – 1.29 (m, 4H), 1.08 – 0.82 (m, 3H).

methoxyphenyl)pentyl)carbamate ¹H NMR (300 MHz, CDCl₃) δ

7.23 (t, J = 7.8 Hz, 1H), 7.01 – 6.74 (m, 3H), 5.56 (br, 1H), 4.60

(br, 1H), 3.95 – 3.63 (m, 4H), 2.11 – 1.85 (m, 1H), 1.67 – 1.02 (m,

13H), 0.86 (t, J = 7.0 Hz, 3H).

(1R,2S)-1-Amino-1-phenylpentan-2-ol (16c). Title compound

was obtained following the procedure described for 16a. to afford

title compound as a white amorphous solid (66% yield over two

steps). tert-Butyl ((1R,2S)-2-hydroxy-1-phenylpentyl)carbamate

¹H NMR (300 MHz, CDCl₃) δ 7.51 – 7.19 (m, 5H), 5.44 (br, 1H),

4.63 (br, 1H), 3.88 (d, J = 8.8 Hz, 1H), 1.72 – 1.02 (m, 14H), 0.88

(t, J = 7.1 Hz, 3H).

(1R,2S)-1-Azido-1-(3-methoxyphenyl)propan-2-ol (15a). To a

stirred solution of cyclic sulfite 14a (150 mg, 0.66 mmol) in DMF

(5 mL) was added sodium azide (171 mg, 263 mmol). The

reaction flask was then heated to 80 C for 8 h. After completion,

the reaction mixture was poured in 25 mL of water, and then

extracted three times with ether. The combined organic layers

were washed with brine, dried over anhydrous sodium sulfate,

filtered and concentrated under reduced pressure. The crude

azidoalcohol was then purified by silica gel flash chromatography

(n-hexane-EtOAc 9:1) to provide the title compound as a light

yellow oil (88% yield). ¹H NMR (300 MHz, CDCl₃) δ 7.41 – 7.23

(m, 1H), 6.98 – 6.81 (m, 3H), 4.43 (d, J = 5.7 Hz, 1H), 4.06 – 3.85

(m, 1H), 3.81 (s, 3H), 2.03 (br, 1H), 1.17 (d, J = 6.3 Hz, 3H).

(tert-Butyl

((2S,3R)-3-hydroxy-4-(((1R,2S)-2-hydroxy-1-(3-

methoxyphenyl)propyl)amino)-1-phenylbutan-2-

yl)carbamate (17a). Compound 16a (0.565 mmol) was dissolved

in i-PrOH (7 mL) and 9 (74 mg, 0.283 mmol) was added. The

reaction mixture was stirred at 80 °C for 14 h. The solvent was

evaporated under reduced pressure and the residue was purified

by silica gel flash chromatography (CH₂Cl₂/MeOH 100:0 to 98:2)

providing title compound as an amorphous white solid (65% yield).

¹H NMR (300 MHz, CDCl₃) δ 7.43 – 7.10 (m, 5H), 7.00 – 6.74 (m,

3H), 4.85 (d, J = 8.9 Hz, 1H), 4.38 – 4.23 (m, 1H), 4.12 – 3.95 (m,

2H), 3.93 – 3.75 (m, 4H), 3.75 – 3.61 (m, 5H), 3.62 – 3.48 (m, 2H),

3.47 – 3.15 (m, 1H), 3.04 – 2.48 (m, 6H), 1.48 – 1.12 (m, 9H),

1.05 (d, J = 6.2 Hz, 3H).

(1R,2S)-1-Azido-1-(3-methoxyphenyl)pentan-2-ol (15b). Title

compound was obtained following the procedure described for

15a. The residue was purified by silica gel flash chromatography

(n-hexane-EtOAc 9:1) to afford title compound as a brown oil

tert-Butyl

((2S,3R)-3-hydroxy-4-(((1R,2S)-2-hydroxy-1-(3-

1

(74% yield). H NMR (300 MHz, CDCl₃) δ 7.42 – 7.23 (m, 2H),

methoxyphenyl)pentyl)amino)-1-phenylbutan-2-

7.04 – 6.83 (m, 2H), 4.45 (d, J = 5.8 Hz, 1H), 3.93 – 3.71 (m, 4H),

1.77 – 1.20 (m, 5H), 0.91 (t, J = 6.5 Hz, 3H).

yl)carbamate (17b). Title compound was obtained following the

procedure described for 17a. The residue was purified by silica

gel flash chromatography (CH₂Cl₂/MeOH 100:0 to 98:2) to afford

title compound as a pale yellow oil (71% yield). 1H NMR ¹H NMR

(300 MHz, CDCl₃) δ 7.39 – 7.07 (m, 5H), 6.95 – 6.72 (m, 3H), 4.74

(d, J = 8.8 Hz, 1H), 3.90 – 3.74 (m, 5H), 3.66 – 3.36 (m, 3H), 3.13

– 2.74 (m, 6H), 2.74 – 2.51 (m, 2H), 1.55 – 1.12 (m, 13H), 0.87 (t,

J = 7.0 Hz, 3H).

(1R,2S)-1-Azido-1-phenylpentan-2-ol (15c). Title compound

was obtained following the procedure described for 15a. The

residue was purified by silica gel flash chromatography (n-

hexane-EtOAc 9:1) to afford title compound as a brown oil (74%

yield). ¹H NMR (300 MHz, CDCl₃) δ 7.58 – 7.29 (m, 4H), 4.48 (d,

J = 5.7 Hz, 1H), 3.79 (d, J = 5.7 Hz, 1H), 1.76 – 1.17 (m, 5H), 1.06

– 0.83 (m, 3H).

tert-Butyl

((2S,3R)-3-hydroxy-4-(((1R,2S)-2-hydroxy-1-

(17c).

(1R,2S)-1-Amino-1-(3-methoxyphenyl)propan-2-ol (16a). To a

solution of 15a (120 mg, 0.58 mmol) and di-tert-butyldicarbonate

(164 mg, 0.75 mmol) in EtOAc (5 mL), 10% Pd/C (15 mg) was

added and the reaction mixture was stirred at 25 °C under

hydrogen atmosphere for 16 h. The reaction mixture was then

filtered through a celite plug. The residue containing tert-butyl

phenylpentyl)amino)-1-phenylbutan-2-yl)carbamate

Title compound was obtained following the procedure described

for 17a. The residue was purified by silica gel flash

chromatography (CH₂Cl₂/MeOH 100:0 to 98:2) to afford title

1

compound as a pale yellow oil (71% yield). H NMR (300 MHz,

CDCl₃) δ 7.50 – 7.09 (m, 10H), 4.77 (d, J = 8.8 Hz, 1H), 3.95 –

3.78 (m, 2H), 3.78 – 3.33 (m, 2H), 3.31 – 3.00 (m, 2H), 2.98 –

2.74 (m, 2H), 2.76 – 2.51 (m, 2H), 1.65 – 1.06 (m, 13H), 0.86 (t, J

= 7.0 Hz, 3H).

((1R,2S)-2-hydroxy-1-(4-methoxyphenyl)propyl)carbamate

¹H

NMR (300 MHz, CDCl₃) δ 7.26-7.20 (m, 1H), 6.85-6.75 (m, 3H),

5.51 (br, 1H), 4.56 (br, 1H), 4.10-3.96 (m, 1H), 3.76 (s, 3H), 2.31

(d, J = 6.6 Hz, 1H), 1.39 (s, 9H), 1.05 (d, J = 6.6 Hz, 3H) was

submitted to the following step without further purification. The

compound was dissolved in CH₂Cl₂(2 mL) and TFA (300 μL) was

added. The reaction mixture was stirred at 25 °C for 2 h, then

solvents evaporated. CH₂Cl₂(3 mL) was added to the residue and

tert-Butyl

methyl(1-(4-methyloxazol-2-yl)ethyl)carbamate

(22). A solution of 21 (110 mg, 0.486 mmol) in diethyl ether (5 mL)

was cooled to -78 °C and treated with TMEDA (95 µL, 0.63 mmol),

followed by s-BuLi (45 µL, 0.63 mmol) dropwise. The mixture was

14

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FULL PAPER

stirred for 30 min at -78 °C and treated with a solution of

iodomethane (60 µL, 0.97 mmol) in 1 mL of diethyl ether and then

slowly warmed to room temperature and stirred for 8 h. The

mixture was diluted with 6 mL of water and extracted with ether (2

x 20 mL) and washed with saturated aqueous NH₄Cl solution. The

combined extracts were dried over Na₂SO₄, filtered and

concentrated under reduced pressure to give a crude product

which was purified by column chromatography over silica gel to

afford 22 (64 mg, 55%) as a colorless oil and 28 mg of starting

material was recovered. ¹H NMR (400 MHz, CDCl₃): δ 7.30 (q, J

= 4.0 Hz, 1H), 5.54 (m, 0.6H), 5.20 (m, 0.4H), 2.67 (brs, 3H), 2.15

(d, J = 1.2 Hz, 3H), 1.54 (d, J = 7.0 Hz, 3H), 1.46 (s, 9H). LRMS-

ESI (m/z): 241 [M+H]⁺.

132.2, 131.2, 130.1, 129.9, 125.7, 125.3, 52.4, 47.2, 32.8, 29.58,

28.2, 21.1, 16.1, 14.9, 11.1. LRMS-ESI (m/z): 303 [M+H] ⁺.

Acknowledgements

Financial support by the National Institutes of Health and Purdue

University is gratefully acknowledged. A.D.M also wishes to

acknowledge partial support from the Walther Cancer Foundation.

NMR, mass spectrometry and protein crystallization were

supported in part by the Purdue Center for Cancer Research

Shared Resources, which are supported by NIH grant (P30

CA023168). Use of the Advanced Photon Source, an Office of

Science User Facility operated for the U.S. Department of Energy

(DOE) Office of Science by Argonne National Laboratory, was

supported by the U.S. DOE under Contract No. DE-AC02-

06CH11357. Use of the LS-CAT Sector 21 was supported by the

Michigan Economic Development Corporation and the Michigan

Technology Tri-Corridor (Grant 085P1000817).

3-Methyl-5-(methyl(1-(4-methyloxazol-2-

yl)ethyl)carbamoyl)benzoic acid (24). Methyl 3-methyl-5-

(methyl(1-(4-methyloxazol-2-yl)ethyl)carbamoyl)benzoate. To a

stirred solution of 22 (64 mg, 0.26 mmol) in dichloromethane (3

mL) was added TFA (1 mL) at 0 ^oC under argon atmosphere and

the mixture was stirred at 23 ^oC for 2 h. Upon completion,

trifluoroacetic acid and CH₂Cl₂were removed under reduced

pressure, the resulting residue was dissolved in ethyl acetate and

washed with saturated aqueous NaHCO₃solution. The organic

layer was dried over anhydrous Na₂SO₄, filtered and concentrated

under reduced pressure to afford amine derivative (40 mg) which

used for next step without further purification.

Abbreviations: BACE1, beta-site amyloid precursor protein

cleaving enzyme 1; BACE2, beta-site amyloid precursor protein

cleaving enzyme 2; DPP-4, dipeptidyl peptidase 4; FDA, Food

and Drug Administration; SAR, structure-activity relationships;

SGLT2, sodium-glucose linked transporter 2.

To a solution above N-methyl-1-(4-methyloxazol-2-yl)ethan-1-

amine (40 mg, 0.28 mmol) and 3-(methoxycarbonyl)-5-

methylbenzoic acid 23 (66 mg, 0.34 mmol) in CH₂Cl₂(4 mL) at

23 °C was added EDCI. HCl (109 mg, 0.57 mmol), HOBt^.H₂O (74

mg, 0.57 mmol) and N,N-DIPEA (150 µL, 0.85 mmol). The

resulting mixture was stirred at 23 °C for 12 h. After this period,

the reaction was quenched with saturated aqueous NaHCO₃

solution. The mixture was extracted with EtOAc, dried over

anhydrous Na₂SO₄, filtered and concentrated under reduced

pressure. The residue was purified by silica gel column

chromatography (40% EtOAc/hexanes) to provide the

corresponding amide derivative Methyl 3-methyl-5-(methyl(1-(4-

methyloxazol-2-yl)ethyl)carbamoyl)benzoate (60 mg, 66% over 2

steps). ¹H NMR (400 MHz, CDCl₃) δ 8.02-7.84 (m, 2H), 7.47 (m,

1H), 7.36 (m, 1H), 6.05 (m, 0.6H), 4.96 (m, 0.4H), 3.88 (s, 3H),

2.86 (s, 1.4H), 2.73 (1.6H), 2.39 (s, 3H), 2.14 (s, 3H), 1.68-1.54

(m, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 171.0, 170.5, 166.4, 162.3,

161.7, 139.0, 138.8, 136.2, 136.1, 135.0, 134.9, 132.1, 131.7,

131.3, 130.3, 130.1, 125.1, 124.8, 52.2, 46.9, 36.5, 32.5, 28.0,

24.6, 21.1, 16.2, 15.0, 11.3. LRMS-ESI (m/z): 317 [M+H] ⁺.

Keywords: BACE2 inhibitors • β-secretase 2 • Tmem27 • type 2

diabetes • structure-based design

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16

10.1002/cmdc.201800725

ChemMedChem

FULL PAPER

Entry for the Table of Contents

Me

OH

H

CF₃

O

N

NH

3l

O

N

Ph

Me

Ph

K_i= 1.6 nM (BACE2)

K_i= 815 nM (BACE1)

Me

3l

An energy-minimized model of

in

BACE2 active site is shown on the the right

Design of Selective BACE2 Inhibitors: Selective BACE2 inhibitors have been designed and synthesized as a potential new

treatment for type 2 diabetes. Utilizing the X-ray structural studies with BACE1 along with computational docking approaches with

BACE2, a series of potent and highly selective BACE2 inhibitors containing hydroxyethylamine isostere have been designed. The

present investigation led to the identification of structural determinants and development for achieving potency and selectivity towards

BACE2 enzyme. Inhibitor 3l exhibited excellent BACE2 potency and high selectivity over BACE1.

17

Article Doi

Highly Selective and Potent Human β-Secretase 2 (BACE2) Inhibitors against Type 2 Diabetes: Design, Synthesis, X-ray Structure and Structure–Activity Relationship Studies

DOI: 10.1002/cmdc.201800725

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Authors:

Article abstract of DOI:10.1002/cmdc.201800725

Full text of DOI:10.1002/cmdc.201800725

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