Generation, optimization and characterization of novel anti-prion compounds

B i o o r g an i c &M e d i c i n a l C h e m i s tr y 2 8 (2 020 )1 15717

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Generation, optimization and characterization of novel anti-prion

compounds

Andrea Altieri^a,b, Evgeny A. Spiridonov^a, Semen I. Sivtzev^a, Daisuke Ishibashi^c, Silvia Biggi^d,

Noriyuki Nishida^c, Emiliano Biasini^d, Alexander V. Kurkin^a,e

^aDepartment of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1–3, Moscow 119991, Russian Federation

^bEDASA Scientiﬁc, Via Stingi 37, San Salvo 66050, Italy

^cDepartment of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan

^dDulbecco Telethon Laboratory of Prions & Amyloids, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Via Sommarive 9,

Trento 38123, Italy

^eA.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, Vavilov St. 28, Moscow, Russian Federation

A R T I C L E I N F O

A B S T R A C T

Keywords:

Prion

Prions are misfolded proteins involved in neurodegenerative diseases of high interest in veterinary and public

health. In this work, we report the chemical space exploration around the anti-prion compound BB 0300674 in

order to gain an understanding of its Structure Activity Relationships (SARs). A series of 43 novel analogues,

based on four diﬀerent chemical clusters, were synthetized and tested against PrP^Scand mutant PrP toxicity

assays. From this biological screening, two compounds (59 and 65) emerged with a 10-fold improvement in anti-

prion activity compared with the initial lead compound, presenting at the same time interesting cell viability.

Lead optimization

Screening

PrP^C

PrP^Sc

1. Introduction

prion protein called PrP^Cto be transformed in an infective abnormal

isoform version called PrP^Sc.¹¹

Following several severe Bovine Spongiform Encephalopathy (BSE,

also known as Mad Cow disease) outbreaks in the UK during

1986–2001,¹with heavy consequences for both the UK bovine meat

market economy and public health, scientiﬁc attention has turned to its

etiological agent: the prions. Prions are misfolded proteins involved in

neurodegenerative diseases in animals such as BSE, scrapie²(in sheep)

and Chronic Wasting Disease (CWD; in deer)³and are also responsible

in humans for Transmissible Spongiform Encephalopathies (TSE).⁴

Human TSEs may be classiﬁed, depending upon nature, in diﬀerent

variations such as Creutzfeldt-Jakob disease (CJD) & the variant

The PrP^C/PrP^Sctransformation mechanism is attributed at the in-

teraction of PrP^Scaggregates with PrP^C, where it acts as a catalytic

element.¹²PrP^Schas a propensity to self-propagate by engaging with

PrP^Cmolecules on the cell surface, and forcing their misfolding and

incorporation into growing ﬁbrils.^4,10–12

The misfolding prion-like mechanisms have postulated also in the

brain neurodegeneration caused by Alzheimer and Parkinson dis-

eases.¹³

It should be noted that despite PrP^Screpresent a putative biological

target for blocking the neurodegenerative disorders, unfortunately this

misfolded protein is not an easy target since its composition is not

uniform and its protein may exist in diﬀerent conformations know as

prion strains.^14,15

Creutzfeldt-Jakob

disease

(vCJD),^4,5

kuru,⁶

Gerstmann–-

Sträussler–Scheinker syndrome (GSS)⁷and Fatal Familia Insomnia

(FFI).⁸

Some statistical correlations were found pointing out that vCJD can

be transmitted to humans by the consumption of BSE aﬀected bovine

meat.^4,9Moreover, some concern was raised about possible human to

human transmission, too.⁴Since BSE also proved to be statistically re-

lated to the consumption of “bone & meat” feeder to farmed animals,

this kind of feed supplement has been strongly restricted or forbidden.⁴

The molecular pathology of TSE is currently based on what is called

the prion hypothesis.^4,10This hypothesis is based on the capability of a

Interestingly, there have been reports of small molecule compounds

capable of interfering with the misfolding process. These compounds

range from congo red to Cu²⁺chelators (Fig. 1).^4,14,16–19Indeed, prion

proteins have been reported to bind strongly to Cu²⁺and, probably,

this ion plays a role as a cofactor in the replication process.¹⁸

However, it should be noted that none of the anti-prion compounds,

so far identiﬁed, have reported good eﬀects in-vivo. This, may be at-

tributed to the diﬀerent prion strain conformers and to the possibility

E-mail addresses: aaltieri@edasascientiﬁc.com (A. Altieri), dishi@nagasaki-u.ac.jp (D. Ishibashi), emiliano.biasini@unint.it (E. Biasini),

kurkin@direction.chem.msu.ru (A.V. Kurkin).

https://doi.org/10.1016/j.bmc.2020.115717

Received 15 May 2020; Received in revised form 4 August 2020; Accepted 13 August 2020

A v a i l a b l e o n l i n e 2 5 A u gu s t 2 020

A. Altieri, et al.

Bioorganic&MedicinalChemistry28(2020)115717

Fig. 2. Compound 6 chemical structure with anti-prion activity on N2a-22L and

N2a-RML prion strains along its calculated physical chemistry properties via

JChem v 8.8.0 ChemAxon software. In the chemical structure have been

highlighted four diﬀerent areas A-D were the chemical modiﬁcations have

performed in order to improve its performances against prion infected cells.

Fig. 1. Example of compounds with reported anti-prion activities. Compounds

3 and 4 are cation chelators.

that other elements may play a role in the misfolding prion conver-

sion.¹⁴

Another key challenge, in this area, is that that the process take

place in the Central Nervous System (CNS), therefore any prion mis-

folding inhibitors should be capable of reaching and passing the Blood

Brain Barrier (BBB), which is extremely selective and as for small mo-

lecules is recommendable a small molecular weight along a small polar

surface area with correct lipophilicity balance and ultimately low

content of hydrogen bonding acceptors (HBA) elements.²⁰

Table 1

Cluster A PrP^Scscreening results.^a

Compound ID

PrP^Scratio (%)

(

) 6

85.8

100.2

84.8

83.7

102.6

95.1

74.5

80.7

75.7

77.5

93.7

32.1

(1R,2S)-6

17.9

16, BB 0310805

17, BB 0310810

18, BB 0323010

19, BB 0323009

20, BB 0323008

21, BB 0323007

22, BB 0323016

23, BB 0323018

24, BB 0,323,011

10.9

35.3

In light of the opportunity for a drug discovery project based on

small molecules acting as prion interfering agents we decided to focus

on the optimization of fragment-like compounds 6 (Fig. 2), which had

already showed interesting anti-prion activity on the PrP^Scstrains N2a-

RML (more active) and N2a-22L (less active).²¹Its small molecular

weight (MW 305) and other physical chemistry properties makes it an

ideal candidate for passing the BBB barrier (small drug CNS compounds

tend to have on average MW of 400–500 Da, TPSA of 50–60, HBD 1.5,

HBA 2, pKa 4–10)¹⁹this on to of a good tox proﬁle (CC₅₀).²¹Moreover,

initial hits characterized by a small MW tend to be more proﬁcient in

the follow-up chemical optimization stages. In fact, by having a MW

smaller than traditional High Throughput Screening (HTS) hits (around

450–500 Da MW) these compounds presents a better Ligand Eﬃcacy

(LE), intended as the ratio of the aﬃnity of a ligand divided by the

number of heavy atoms in the molecule²²and, therefore, there is more

room for chemically growing by adding and exploring additional hy-

drogen bond (HB) moieties without rendering the optimized compound

too lipophilic or insoluble, which is a must for good per os formula-

tions.²³In order to gather a prion strain selectivity idea, and since

compound 6 was already proﬁled for N2a-22L and N2a-RML PrPSc

strains,²¹and due to current FK PrPSc strain availability in Japan, we

opted in proﬁling compound 6 and the new series versus PrPSc N2a-FK

strain.

22.2

24.6

11.1

10.3

24.7

47.36

20.2

a

PrP^Scwere detected from 10 µM compound-treated

N2a-FK cells. Statistics: each PrP^Scratio (%) was compared

with a solvent-treated group as control (100%, Tukey-

kramer) and the mean

corded.

standard deviation (SD) was re-

(Cluster B), then modiﬁcations of the cyclohexyl ring (Cluster C) and

modiﬁcations of the benzylamine moiety (Cluster D).

For each cluster, we generated a virtual set of chemically feasible

compounds, based on commercially available reagents. Each virtual

tangible library from each cluster (A–D) was prioritized for synthesis

based on previous screening results¹⁵and by considering the BBB per-

meability, too. For the BBB prioritizations, these four clusters of virtual

compounds were ﬁltered according to the following predictive BBB

model:^{19–26 1}

ClogP (N

+

O)

>

0

TPSA

<

60

2. Results and discussion

2.1. Compounds from Cluster A

Since 6 presented two chiral centers and the anti-prion activity was

veriﬁed on only the racemate of the trans-amino alcohol 6, as a very

ﬁrst step, via a Shi enantioselective synthesis,²⁴its (1R,2S) enantiomer

was achieved with a 95% ee purity (characterization reported in the

chemistry paragraph) and proﬁled against PrP^SCinhibitory activity

using a N2a-FK ells mouse persistently infected with an FK-1 prion

strain, as per a previously described method.²⁵

Compounds 16–24 (Scheme 1) were synthetized from their corre-

sponding arylacetylenes, using a literature described procedure²¹and

proﬁled against PrP^Scfollowing a literature described method, too.²⁵

Statics against PrP^Scratio (%) performed versus control using Tukey-

Kramer multiple comparison test. Their recovery PrP^Scratios (%) are

listed in Table 1.

However, the (1R,2S) 6 enantiomer presented equipotent anti-PrP^Sc

activity comparable to its racemic form (Table 1) and, therefore, no

further eﬀorts were made to produce and isolate the other enantiomer.

Therefore, we instead focused on expanding the chemical space

around 6. Based on its synthesis,²¹and in order to explore and build a

Structure-Activity Relationship (SAR) model, we envisioned four pos-

sible modiﬁcations of 6: one around the phenyl ring next to the acet-

ylene group (Cluster A), a second set of modiﬁcations at the triple bond

Notably, compound 6, which had good anti-prion activity versus

N2a-22L and N2a-RML strains,²¹reported no statistically relevant eﬀect

on N2a-FK cells. Also, from the Cluster A screening results, there ap-

pears not to be any signiﬁcant anti-prion activity in the whole series.

The only compound, which seem to perform slightly better, is 20 (m-

¹Predictive BBB permeability data available from the SI.

2

A. Altieri, et al.

Bioorganic&MedicinalChemistry28(2020)115717

Scheme 3. Compound 23 synthetic pathway.²⁷

Scheme 1. Chemical structures of the compounds belonging to Cluster A and

their synthetic approach.

Scheme 4. Compound 37 - synthetic pathway.

activity solely versus N2a-RML and N2a-22L strains, since 26 was in-

eﬀective in N2a-RML and N2a-22L cells, as opposed to the results for

6.²¹

2.3. Compounds from Cluster C

In order to enlarge the SARs around the cyclohexyl moiety of

compound 6, based on pragmatic synthetic pathways (Schemes 3–6),

six additional compounds were evaluated (33, 37, 44–47 ).

Among them, the open ring form of 6, compound 33, was synthe-

tized as depicted in Scheme 3, via the epoxidation of 31 followed by the

epoxide 32 ring opening via the addition of benzylamine in water.

Compound 33′s anti-prion activity turned out to be of low interest

(Table 3) and therefore no other eﬀorts to synthetize open ring deri-

vatives were pursued.

Scheme 2. Compounds from Cluster B and corresponding synthetic scheme for

25–29.

Fluoro-Ph substituted). Interestingly, replacing the F of the metha po-

sition with a methyl group (such in compound 24) or with a MeOC(O)-

moiety (such in compound 17) leads to no activity.

After exploring the ring’s open form, attention shifted to the ring’s

enlarged analogue 37, which was synthetized using the same synthetic

route as per 6, but replacing cyclohexanone with cycloheptanone at the

initial step, as depicted in Scheme 4. However, this modiﬁcation pro-

duced a compound, which was not relevant in terms of activity.

To this end, two cyclohexane ring isoster analogues were produced

using the synthetic scheme as depicted by Scheme 5.

2.2. Compounds from Cluster B

Playing with the reduction of the compound 6 triple bond (Scheme

2), it was possible to evaluate the SARs for an additional three com-

pounds 25, 28 and 29 (Cluster B). Starting from 6, under mild reduction

conditions it was possible to achieve compound 25, while further

pushing the reduction conditions led to cleavage of the benzyl moiety

and to the generation of the free amine 27. Compound 27 was exploited

for generating the corresponding benzoyl and phenylsulfonyl deriva-

tives (28–29) in order to evaluate their biological behavior, too.

Alongside them, we evaluated a previously made compound, 26,²¹

which presented the trans double bond moiety instead of the triple

bond. None of these compounds manifested an interesting biological

anti-prion response (Table 2). These results indicate that replacing the

triple bond with double or simple bonds did not render the compounds

active. We can speculate that rigidity at that position is beneﬁcial for

It should be noted that the Boc cleavage of compound 45 turned out

to be somewhat problematic due to the low stability of the propargyl

alcohol 45 under acidic conditions. This resulted in a complex and in-

separable mixture. Considering that neither 44 nor 45 proved to be

active against prions (Table 3), no other eﬀorts were performed in this

direction.

An attempt to gather a conformationally constrained analogue of 6

was made by treating 6 with 1,1-carbonyldiimidazole (CDI) and 4-

(dimethylamino)pyridine (DMAP). This produced a crude product

containing a mixture of compounds 46 and 47 (Scheme 6), which was

Table 2

Cluster B PrP^Scscreening results.^a

Compound ID

PrP^Scratio (%)

26, BB 0300668²¹

27, BB 0310495

28, BB 0310746

29, BB 0310747

81.6

30.4

23.3

88.0

126.3

112.0

64.8

69.7

a

PrP^Scwere detected from 10 µM compound-treated

N2a-FK cells. Statistics: each PrP^Scratio (%) was compared

with a solvent-treated group as control (100%, Tukey-

kramer) and the mean

corded.

standard deviation (SD) was re-

Scheme 5. Compounds 41, 44–45 - synthetic pathway.

3

A. Altieri, et al.

Bioorganic&MedicinalChemistry28(2020)115717

Table 4

Cluster D PrP^Scscreening results.^a

Compound ID

PrP^Scratio (%)

R₁

H

R₂

48, BB 0305177

58.8

61.2

68.6

83.3

96.1

75.5

84.8

101.9

25.7

17.1

2.0

49, BB 0305171

50, BB 0305178

51, BB 0310741

52, BB 0310736

53, BB 0310738

54, BB 0310737

55, BB 0305182

H

Scheme 6. Compounds 46–47 synthetic pathway.

Table 3

18.4

20.7

18.6

25.9

Cluster C PrP^Scscreening results.^a

Compound ID

PrP^SCratio (%)

33, BB 0305187

37, BB 0310748

44, BB 0263237

45, BB 0311012

46, BB 0310744

47, BB 0310745

120.1

75.7

85.2

108.4

68.4

96.7

13.7

24.7

15.9

49.5

23.0

36.4

a

36.6

PrP^Scwere detected from 10 µM compound-treated

N2a-FK cells. Statistics: each PrP^Scratio (%) was compared

with a solvent-treated group as control (100%, Tukey-

kramer) and the mean

recorded.

standard deviation (SD) was

56, BB 0310739

57, BB 0310652

58, BB 0310742

63.4

116.0

86.0

32.5

H

eventually separated and both compounds were successfully isolated

and characterized. However, both compounds performed poorly on the

anti-prion assay (Table 3).

23.6

2.4. Compounds from Cluster D

22.6

Finally, we explored the anti-prion activities of a set of 6 analogues

with modiﬁcations of the substituents at the nitrogen atom. The results

are reported in Table 4. Compounds 48–62 and 64 were obtained by

treating the epoxide 1-(2-phenylethynyl)-7-oxabicyclo[4.1.0]heptane

with the corresponding amine as per a general procedure previously

reported in the literature.²¹Compounds 65 and 60 were instead derived

from slightly diﬀerent epoxides (the 1-(2-p-methyphenylethynyl)-7-

oxabicyclo[4.1.0]heptane and the 1-(2-p-ﬂuorophenylethynyl)-7-ox-

abicyclo[4.1.0]heptane, respectively) against the corresponding amines

as reported in the chemistry experimental part. Compound 63 was in-

stead obtained by direct alkylation of BB 0300674 (Scheme 7). 63 was

meant as a precursor of 58, however, internal cyclization of 63 proved

to be problematic due to cleavage of the benzyl group.

59, BB 0305179

60, BB 0323017

61, BB 0323014

32.7

75.2

77.5

19.8^**

20.9

H

Me

H

47.36

62, BB 0310740

63, BB 0310743

102.6

76.1

6.6

H

29.1

The results obtained from this cluster were very promising and

compounds 59 and 65 presented an interesting anti-prion recovery rate.

It is interesting to note that in the entire Cluster D series none of the

compounds except for two, 59 and 65, proved to be relevant in terms of

anti-prion activity. Compounds 59 and 65, both have a cyclohexyl

group as a replacement for the phenyl group in 6, and 65 present a para

F atom in its phenyl ring. The cyclohexyl ring is larger and more ﬂexible

than the phenyl ring; these rings also have distinct electronic organi-

zation. Those results suggest that volume, ﬂexibility, and electronic

organization at that position have a signiﬁcant impact on activity.

Additionally, the molecule 65 has a para-ﬂuorine atom in the phenyl

ring next to the acetylene group as a replacement for the hydrogen

atom in 6. These bioisosteres (H and F) resulted in very similar po-

tencies for 59 and 65. By comparison with 60, which bears the slightly

larger methyl group, it is possible to conclude that a small substituent at

that position is beneﬁcial for activity. Notably, the substituent R₂in 59

is much more ﬂexible and can occupy a larger chemical space than the

R₂substituent in 64 (which can be seen as conformationally restricted

64, BB 0323019

66.7

30.1

H

65, BB 0323015

66, BB 0323013

a

35.5

54.8

17.1^***

33.0

F

H

PrP^Scwere detected from 10 µM compound-treated N2a-FK cells. Statistics:

each PrP^Scratio (%) was compared with a solvent-treated group as control

(100%, Tukey-kramer) and the mean

** p < 0.01.

standard deviation (SD) was recorded.

*** p < 0.001 vs control.

4

A. Altieri, et al.

Bioorganic&MedicinalChemistry28(2020)115717

Scheme 7. Compounds 63 synthetic pathway.

Fig. 4. Assay performed on HEK293 cells expressing ΔCR PrP. The bar graphs

show the activity of the indicated molecules tested by DBCA. Data report the %

of cell viability rescue, calculated using the following equation: R = (T − Z)/

(U − Z) (R: rescuing eﬀect; T: cell viability in compound-treated samples; Z: cell

viability in Zeocin-treated samples; U: cell viability in untreated samples).

Statistically-signiﬁcant diﬀerences (*) between compound-treated and un-

treated cells were estimated by a One-way Anova, Dunnett post-hoc test (p

values are indicated as * < 0.001). Rescuing dose at 50% (RD₅₀) values (ex-

Scheme 8. Synthetic pathway for compounds 48–66. R₁and R₂groups as per

Table 4.

version of 59), showing that ﬂexibility at that position must be a key

feature for activity.

pressed in µM) were as follow: 6 (BB 0300674), RD₅₀= 3.435

2.2, 59 (BB

3. Biology

0305179), RD₅₀= 0.166

0.141, 24 (BB 0323011), RD₅₀= 2.081 0.137,

65 (BB 0323015), RD₅₀= 0.523

0.0751.

3.1. Biological characterization in cell-based models of prion replication

and cell toxicity

diﬀerent concentrations, or were exposed to vehicle control (DMSO).

Cell viability was then monitored by MTT assay (Fig. 4). The results

revealed a rescuing activity for all the molecules tested, although

compound 59 (BB 0305179) was found to be the most potent among

the four molecules, with a dose-dependent, signiﬁcant eﬀect already

observed at 0.1 µM. Of note, molecules 59 (BB 0305179) and 65 (BB

0323015) showed decreased activity at the highest concentration

(30 µM), likely reﬂecting an intrinsic cytotoxicity. Conversely, none of

the compounds showed cytotoxicity at any concentration below 10 µM

(Fig. 5) when tested in HEK293 cells in the absence of Zeocin.

To this end, the IC₅₀measurement was performed on the PrP^Scassay

for the best two performing compounds, 59 and 65 (Fig. 3).

As per the PrP^Screcorded recovering data, compounds 59 and 65

resulted in comparable IC₅₀of 4.7

1.9 and 5.6

1.4 µM, respec-

tively. Multiple previous reports have shown that PrP molecules car-

rying disease-associated or artiﬁcial mutations in the central region of

the protein sharply increase the sensitivity of a number of cells to the

toxicity of cationic antibiotics such as aminoglycosides or phleomycin

derivatives (e.g. G418 and Zeocin, respectively). This phenotype has

been used to establish a so-called drug-based cellular assay (DBCA)

previously employed to study the toxicity of mutant PrP molecules.^21,28

Here, we employed the DBCA to test the activity of selected com-

pounds 6, 24, 59 and 65 to inhibit the toxicity of a speciﬁc PrP mutant

carrying a deletion in the hydrophobic domain (residues 105–125, so

called ΔCR PrP). HEK293 cells stably expressing mutant PrP were ex-

posed for 24 h to Zeocin in the presence of each individual compound at

Fig. 3. PrP^Scassay was performed using N2a-FK cells. Compounds 59 and 65,

PrP^Scintensity bands. To calculate IC₅₀, the PrP^Scband intensity from WB was

plotted for each concentration compound (1, 5 and 10 µM) and ﬁtted to the

Boltzman sigmoidal equation using Origin 8.5 software. For statistical data

Fig. 5. Assay performed on HEK293 cells. The bar graphs report the intrinsic

cytotoxicity of the diﬀerent molecules, estimated by MTT assay, and expressed

as % of cell viability in vehicle controls. Statistically-signiﬁcant diﬀerences (*)

between compound-treated and untreated cells were estimated by One-way

Anova, Dunnet post-hoc test (p values are indicated as * < 0.01). Lethal dose

at 50% (LD₅₀) values were > 30 µM for all the compounds.

analysis, the mean

was considered.

SD obtained from the results of at least three experiments

5

A. Altieri, et al.

Bioorganic&MedicinalChemistry28(2020)115717

3.2. Biological assays experimental protocols

4.2. Synthetic protocols and compound characterizations

The PrP^Scassay was performed as described previously in litera-

ture.²⁵N2a-FK cells were seeded at 2.5 × 10⁵cells per well in a 12-well

plate and culture at 37 °C for O/N in a CO₂incubator. The next day, the

compounds (10 µM) were treated to the cells and culture at 37 °C in the

CO₂incubator. After 48 h, the treated cells were collected lysed. To

detect PrP^Sc, the lysate was treated with 20 μg/mL proteinase K at 37 °C

for 30 min. The PK-treated samples were electrophoresed with 15%

SDS-PAGE, and transferred from the gel to PVDF membrane. In im-

munoblotting, anti-PrP Ab (M20, SantaCruz Biotechnology) and anti-

goat IgG-HRP (Jackson ImmunoResearch Laboratories) were used. The

bands were detected by Clarity ECL (Bio-Rad). The band intensities

were quantiﬁed using ImageJ software (NIH). The statistical sig-

niﬁcance in the result was determined using the unpaired Student’s t

test. The results represent at least three independent experiments.

4.2.1. General procedure for epoxide ring opening reaction via benzylamine

(GP-I)²¹(Scheme 1)

To a vigorously stirred solution of aryl oxirane (1 equiv) and ben-

zylamine (2 equiv) in acetonitrile (0.5–1 M solution of epoxide) lithium

perchlorate (2 equiv) was added in one portion. The reaction mixture

was stirred at 50–60 °C until the full consumption of the starting ep-

oxide was achieved (TLC control, typically 12–20 h). The reaction

mixture was cooled to an ambient temperature and poured into 2 vo-

lumes of water followed by the extraction with 2 to 3 times (half of the

reaction mixture volume each time) of dichloromethane. Combined

organic extracts were dried over anhydrous sodium sulfate and con-

centrated under reduced pressure on a rotary evaporator. The residue

was puriﬁed by ﬂash chromatography (eluting with hexane – EtOAc or

CH₂Cl₂– MeOH in variable proportions) to aﬀord amino propargylic

alcohols 16–24 as dark yellow/orange oils or an oﬀ-white solid in

28–63% yields.

3.3. DBCA and cytotoxicity proﬁling

4.2.1.1. Methyl 4-[2-[(1RS,2SR)-2-(benzylamino)-1-hydroxy-cyclohexyl]

ethynyl]benzoate (16, BB 0310805). Compound 16 was synthesized

according to the general procedure GP-I from Methyl 4-[2-(7-

oxabicyclo[4.1.0]heptan-1-yl)ethynyl]benzoate (7) (2.18 g, 8.5 mmol,

1 eq) and benzylamine (1.86 mL, 17 mmol, 2 eq) in MeCN (10 mL, 1 M),

stirring the reaction mixture at 60 °C for 12 h and isolated after ﬂash

chromatographic puriﬁcation (hexane – EtOAc, from 20:1 to 10:1) in

the amount of 1.69 g (54%) as a yellow oil.

The DBCA was performed as described previously, with minor

modiﬁcations.²¹Brieﬂy, HEK293 cells (ATCC, CRL-1573) expressing

ΔCR PrP were seeded on 24-well plates at approximately ∼ 60% con-

ﬂuence. On day 2, the cells were treated with diﬀerent concentrations

of each compound or vehicle control and after 6 h Zeocin was added at

a ﬁnal concentration of 400 μg/mL. Intrinsic toxicity of the diﬀerent

molecules was also evaluated using the same experimental conditions

without adding Zeocin. After 24 h, the medium was removed, and the

cells were incubated with 1 mg/mL of 3-(4.5-dimethylthiazol-2-yl)-2.5-

diphenyltetrazolium bromide (MTT, Sigma Aldrich) in PBS for 20 min

at 37 °C. After carefully removing the MTT solution, the formazan

crystals were resuspended in 300 µL of DMSO, and cell viability was

measured with a spectrophotometer (Tecan) by detecting absorbance at

570 nm.

R_f= 0.40 (hexane – EtOAc, 10:1).

¹H NMR: (CDCl₃, 400 MHz) δ = 1.26–1.37 (m, 3H) 1.55 (td,

J = 12.23, 3.91 Hz, 1H) 1.61–1.86 (m, 3H) 2.18–2.27 (m, 2H)

2.42–2.49 (m, 1H) 3.74 (d, J = 13.08 Hz, 1H) 3.92 (s, 3H) 4.04 (d,

J = 12.96 Hz, 1H) 4.45 (br. s., 1H) 7.25–7.30 (m, 1H) 7.32–7.40 (m,

4H) 7.46–7.51 (m, 2H) 7.93–8.01 (m, 2H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 23.6, 26.1, 31.0, 39.9, 51.5, 54.2,

65.5, 72.2, 84.2, 95.3, 121.1, 123.2, 127.2, 128.2, 128.3, 130.5, 131.2,

131.7, 134.4, 140.1, 166.43

4. Chemistry

4.1. Reagents and materials

IR ν_max(KBr): 3437 (br.), 2939, 2856, 1714, 1604, 1441, 1288,

1277, 1115, 1093, 769, 742, 696 cm⁻¹

Reagents were purchased at the highest commercial quality and

used without further puriﬁcation unless otherwise stated. All starting

aryl oxiranes (7–15), 2-(Benzylamino)-1-(phenylethynyl)cyclohexanol

were kindly provided by EDASA Scientiﬁc. Reactions were monitored

by thin-layer chromatography (TLC) carried out on Merck TLC Silica gel

plates (60F₂₅₄), using UV light for visualization and basic aqueous po-

tassium permanganate or iodine fumes as developing agents.

HRMS (ESI) m/z: calculated for C₂₃H₂₆NO₃[M + H]⁺364.1907,

found 364.1915.

4.2.1.2. Methyl 3-[2-[(1RS,2SR)-2-(benzylamino)-1-hydroxy-cyclohexyl]

ethynyl]benzoate (17, BB 0310810). Compound 17 was synthesized

according to the general procedure GP-I from Methyl 3-[2-(7-

oxabicyclo[4.1.0]heptan-1-yl)ethynyl]benzoate

(8)

(3.48 g,

¹H and ¹³C NMR spectra were recorded on Bruker Avance 400 in-

strument with operating frequency of 400 and 100 MHz respectively

13.5 mmol, 1 eq) and benzylamine (2.97 mL, 27.1 mmol, 2 eq) in

MeCN (15 mL, 1 M), stirring the reaction mixture at 60 °C for 12 h

and isolated after ﬂash chromatographic puriﬁcation (hexane – EtOAc,

from 20:1 to 10:1) in the amount of 2.08 g (42%) as a yellow oil.

and

calibrated

using

residual

undeuterated

chloroform

(δH = 7.26 ppm) and CDCl₃(δC = 77.16 ppm), or undeuterated DMSO

(δH = 2.50 ppm) and DMSO‑d₆(δC = 39.51 ppm) as internal refer-

ences. The following abbreviations are used to set multiplicities:

s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet,

m = multiplet, br. = broad.

R_f= 0.50 (Hex:EtOAc, 10:1).

¹H NMR: (CDCl₃, 400 MHz) δ = 1.23–1.37 (m, 4H) 1.49–1.59 (m,

J = 12.35, 12.35, 4.40 Hz, 1H) 1.62–1.87 (m, 3H) 2.16–2.27 (m, 2H)

2.42–2.50 (m, 1H) 3.75 (d, J = 13.08 Hz, 1H) 3.93 (s, 3H) 4.05 (d,

J = 12.96 Hz, 1H) 4.45 (br. s., 1H) 7.32–7.42 (m, 5H) 7.61 (dt,

J = 7.76, 1.38 Hz, 1H) 7.97 (dt, J = 7.83, 1.41 Hz, 1H) 8.10 (t,

J = 1.65 Hz, 1H)

IR spectra were recorded on Thermo Nicolet IR-200 in KBr, nujol or

neat.

High-resolution mass spectra (HRMS) were recorded on Thermo

Scientiﬁc LTQ Orbitrap instrument using nanoelectrospray ionization

(nano-ESI). Low-resolution mass spectra were on Finnigan MAT mass

spectrometer using electron ionization (direct inlet) and an ITD-700

detector with the ionizing electron energy being 70 eV and the mass

range being m/z 35–400.

¹³C NMR: (CDCl₃, 100 MHz) δ = 23.8, 26.9, 30.7, 40.1, 51.5, 54.6,

65.5, 72.2, 84.2, 95.3, 121.1, 123.2, 127.2, 128.2, 128.3, 130.5, 131.2,

131.7, 134.4, 139.3, 164.43

Elemental analysis was performed on EURO EA CHN Elemental

Analyzer. The melting points (m.p.) were measured in open capillaries

and presented without correction.

IR ν_max(KBr): 3454 (br.), 2935, 2858, 1724, 1441, 1298, 1227,

1205, 1105, 910, 754, 733, 700 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₂₃H₂₆NO₃[M + H]⁺364.1907,

6

A. Altieri, et al.

Bioorganic&MedicinalChemistry28(2020)115717

found 364.1910.

J = 7.6 Hz, 1H), 7.28–7.33 (m, 3H), 7.45 (dd, J = 6.7, 3.0 Hz, 2H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 23.4, 25.2, 29.0, 38.3, 50.2, 65.1,

72.3, 86.5, 90.1, 114.2 (d, J = 21.2 Hz), 115.1 (d, J = 21.2 Hz), 122.8,

123.8 (d, J = 2.9 Hz), 128.4, 130.1 (d, J = 8.1 Hz), 131.9, 142.8 (d,

J = 4.4 Hz), 163.1 (d, J = 245.9 Hz).

4.2.1.3. Methyl 2-[2-[(1RS,2SR)-2-(benzylamino)-1-hydroxy-cyclohexyl]

ethynyl]benzoate (18, BB 0323010). Compound 18 was synthesized

according to the general procedure GP-I from Methyl 2-[2-(7-

oxabicyclo[4.1.0]heptan-1-yl)ethynyl]benzoate (9) (2.39 g, 9.3 mmol,

1 eq) and benzylamine (2.04 mL, 19.6 mmol, 2 eq) in MeCN (10 mL,

1 M), stirring the reaction mixture at 60 °C for 12 h and isolated after

ﬂash chromatographic puriﬁcation (hexane – EtOAc, from 20:1 to 10:1)

in the amount of 1.12 g (33%) as a yellow oil.

IR ν_max(KBr): 3446 (br.), 2935, 2858, 1608, 1579, 1485, 1454,

1072, 980, 870, 785, 739, 700, 683 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₂₁H₂₃FNO [M + H]⁺324.1758,

found 324.1761.

4.2.1.6. (1RS,2SR)-2-(Benzylamino)-1-[2-(2-ﬂuorophenyl)ethynyl]

cyclohexanol (21, BB 0323007). Compound 21 was synthesized

according to the general procedure GP-I, from 1-[2-(2-ﬂuorophenyl)

ethynyl]-7-oxabicyclo[4.1.0]heptane (12) (1.18 g, 5.4 mmol, 1 eq) and

benzylamine (1.19 mL, 10.8 mmol, 2 eq) in MeCN (10 mL, 0.5 M),

stirring the reaction mixture at 60 °C for 12 h and isolated after ﬂash

chromatographic puriﬁcation (hexane – EtOAc, from 10:1 to 5:1) in the

amount of 0.69 g (39%) as a yellow oil.

R_f= 0.40 (Hex:EtOAc, 10:1).

¹H NMR: (CDCl₃, 400 MHz) δ = 1.27–1.37 (m, 1H), 1.46–1.58 (m,

2H), 1.69–1.83 (m, 4H), 2.18–2.26 (m, 2H), 2.45–2.49 (dd, J = 11.2,

3.5 Hz, 1H), 3.73 (d, J = 13.1 Hz, 1H), 3.78 (s, 3H), 4.08 (d,

J = 13.1 Hz, 1H), 4.56 (br. s., 1H), 7.24–7.28 (m, 1H), 7.31–7.46 (m,

5H), 7.55–7.63 (m, 2H)

¹³C NMR: (CDCl₃, 100 MHz) δ = 23.6, 26.1, 31.0, 39.9, 51.5, 54.2,

65.5, 72.2, 84.2, 95.3, 121.1, 123.2, 127.2, 128.2, 128.3, 130.5, 131.2,

131.7, 134.4, 140.1, 166.43.

R_f= 0.40 (Hex:EtOAc, 5:1).

IR ν_max(KBr): 3448 (br.), 2935, 2858, 1726, 1446, 1294, 1269,

¹H NMR: (CDCl₃, 400 MHz) δ = 1.19–1.42 (m, 2H), 1.50–1.59 (m,

1H), 1.61–1.85 (m, 3H), 2.11–2.26 (m, 2H), 2.43–2.53 (m, 1H), 3.79

(dd, J = 17.0, 13.4 Hz, 1H), 4.08 (dd, J = 13.4, 9.8 Hz, 1H), 4.40 (br.

s., 1H), 7.00–7.17 (m, 1H), 7.22–7.50 (m, 7H).

1255, 1132, 1082, 758, 737, 700 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₂₃H₂₆NO₃[M + H]⁺364.1907,

found 364.1905.

¹³C NMR: (CDCl₃, 100 MHz) δ = 23.4, 25.2, 28.9, 38.3, 50.6, 65.3,

72.3, 86.4, 90.2, 115.4 (d, J = 21.2 Hz), 124.3 (d, J = 4.1 Hz), 127.4,

128.3, 128.6, 128.6, 128.9 (d, J = 8.0 Hz), 130.4 (d, J = 4.8 Hz), 161.6

(d, J = 245.8 Hz).

4.2.1.4. (1RS,2SR)-2-(Benzylamino)-1-[2-(4-ﬂuorophenyl)ethynyl]

cyclohexanol (19, BB 0323009). Compound 19 was synthesized

according to the general procedure GP-I from 1-[2-(4-ﬂuorophenyl)

ethynyl]-7-oxabicyclo[4.1.0]heptane (10) (1.09 g, 5 mmol, 1 eq) and

benzylamine (1.10 mL, 10 mmol, 2 eq) in MeCN (10 mL, 0.5 M), stirring

the reaction mixture at 60 °C for 12 h and isolated after ﬂash

chromatographic puriﬁcation (hexane – EtOAc, from 10:1 to 5:1) in

the amount of 0.46 g (28%) as a orange oil.

IR ν_max(KBr): 3458 (br.), 2935, 2858, 1493, 1452, 1265, 1101,

1072, 1030, 758, 700 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₂₁H₂₃FNO [M + H]⁺324.1758,

found 324.1761.

4.2.1.7. (1RS,2SR)-2-(Benzylamino)-1-[2-(4-methoxyphenyl)ethynyl]

cyclohexanol (22, BB 0323016). Compound 22 was synthesized

according to the general procedure GP-I from 1-[(4-methoxyphenyl)

ethynyl]-7-oxabicyclo[4.1.0]heptane (13) (1.6 g, 7 mmol, 1 eq) and

benzylamine (1.53 mL, 14 mmol, 2 eq) in MeCN (10 mL, 1 M), stirring

the reaction mixture at 60 °C for 12 h and isolated after ﬂash

chromatographic puriﬁcation (hexane – EtOAc, from 10:1 to 3:1) in

the amount of 1.48 g (63%) as a yellow oil.

R_f= 0.40 (Hex:EtOAc, 5:1).

¹H NMR: (CDCl₃, 400 MHz) δ = 1.21–1.43 (m, 2H), 1.53 (td,

J = 12.2, 4.8 Hz, 1H), 1.62–1.77 (m, 2H), 1.78–1.86 (m, 1H),

2.13–2.26 (m, 2H), 2.44 (dd, J = 11.2, 3.5 Hz, 1H), 3.72 (d,

J = 13.1 Hz, 1H), 4.01 (d, J = 13.1 Hz, 1H), 4.39 (br. s., 1H), 7.03 (t,

J = 8.7 Hz, 2H), 7.28–7.38 (m, 5H), 7.41–7.48 (m, 2H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 23.5, 25.3, 29.0, 38.3, 50.0, 65.0,

72.3, 77.2, 86.4, 90.2, 115.4 (d, J = 21.2 Hz), 122.8, 128.4, 128.4,

129.9 (d, J = 8.1 Hz), 131.8, 135.9, 162.2 (d, J = 245.2 Hz).

IR ν_max(KBr): 3462 (br.), 2933, 2858, 1506, 1454, 1230, 1093, 837,

R_f= 0.40 (Hex:EtOAc, 5:1).

¹H NMR (CDCl₃, 400 MHz) δ = 1.25–1.29 (m, 2H) 1.49–1.59 (m,

1H) 1.60–1.84 (m, 4H) 1.88–2.05 (m, 3H) 2.09–2.17 (m, 1H) 2.48 (br.

s., 1H) 3.80 (s, 3H) 5.35 (dd, J = 7.64, 3.73 Hz, 1H) 6.78–6.83 (m, 2H)

7.28–7.32 (m, 2H) 7.38–7.43 (m, 1H) 7.55 (ddd, J = 8.04, 2.11,

1.10 Hz, 1H) 7.98 (dt, J = 7.82, 1.22 Hz, 1H) 8.06 (t, J = 1.77 Hz, 1H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 23.4, 25.2, 28.9, 38.2, 50.0, 55.3,

64,83, 72.1, 86.3, 90.2, 113.9, 122.8, 128.2, 128.2, 129.4, 131.7,

132.0, 158.8.

739, 700 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₂₁H₂₃FNO [M + H]⁺324.1758,

found 324.1756.

4.2.1.5. (1RS,2SR)-2-(Benzylamino)-1-[2-(3-ﬂuorophenyl)ethynyl]

cyclohexanol (20; BB 0323008).. Compound 20 was synthesized

according to the general procedure GP-I from 1-[2-(3-ﬂuorophenyl)

ethynyl]-7-oxabicyclo[4.1.0]heptane (11) (3.01 g, 13.9 mmol, 1 eq)

and benzylamine (3.04 mL, 27.8 mmol, 2 eq) in MeCN (14 mL, 1 M),

stirring the reaction mixture at 60 °C for 12 h and isolated after ﬂash

chromatographic puriﬁcation (hexane – EtOAc, from 10:1 to 5:1) in the

amount of 2.32 g (51%) as a yellow oil.

IR ν_max(KBr): 3477 (br), 2939, 2861, 1606, 1510, 1442, 1292,

1253, 1129, 1074, 1032, 833, 748 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₂₂H₂₆NO₂[M + H]⁺336.1958,

found 336.1961.

4.2.1.8. (1RS,2SR)-2-(Benzylamino)-1-[2-(4-chlorophenyl)ethynyl]

cyclohexanol (23, BB 0323018). Compound 23 was synthesized

according to the general procedure GP-I, from 1-[2-(4-chlorophenyl)

ethynyl]-7-oxabicyclo[4.1.0]heptane (14) (2.46 g, 10.5 mmol, 1 eq)

and benzylamine (2.01 mL, 21 mmol, 2 eq) in MeCN (11 mL, 1 M),

stirring the reaction mixture at 60 °C for 12 h and isolated after ﬂash

chromatographic puriﬁcation (hexane – EtOAc, from 10:1 to 3:1) in the

amount of 1.21 g (34%) as a yellow oil.

R_f= 0.40 (Hex:EtOAc, 5:1).

¹H NMR: (CDCl₃, 400 MHz) δ = 1.20–1.43 (m, 2H), 1.54 (td,

J = 12.3, 4.8 Hz, 1H), 1.62–1.77 (m, 2H), 1.82 (dd, J = 13.1, 1.8 Hz,

1H), 2.17 (dd, 1H), 2.20–2.27 (m, 1H), 2.45 (dd, J = 11.4, 3.5 Hz, 1H),

3.75 (d, J = 13.4 Hz, 1H), 4.05 (d, J = 13.4 Hz, 1H), 4.35 (br. s., 1H),

6.96 (td, J = 8.4, 2.2 Hz, 1H), 7.10 (d, J = 9.8 Hz, 1H), 7.15 (d,

7

A. Altieri, et al.

Bioorganic&MedicinalChemistry28(2020)115717

R_f= 0.40 (Hex:EtOAc, 5:1).

temperature for 18 h. After this, mixture was ﬁltered through a thin

layer of celite and solvent was removed under reduced pressure to give

0.98 g of desired product.

¹H NMR: (CDCl₃, 400 MHz) δ = 1.23–1.39 (m, 3H) 1.48–1.88 (m,

4H) 2.17–2.26 (m, 2H) 2.45 (dd, J = 10.70, 3.36 Hz, 1H) 3.74 (d,

J = 13.08 Hz, 1H) 4.04 (d, J = 12.96 Hz, 1H) 4.43 (br. s., 1H)

7.25–7.31 (m, 3H) 7.32–7.40 (m, 6H).

R_f= 0.30 (CHCl₃-MeOH-NH₃, 10:1:1). Yield 97%.

¹³C NMR: (CDCl₃, 100 MHz) δ = 23.4, 25.2, 29.0, 38.1, 50.8, 65.1,

72.3, 85.1, 91.4, 121.3, 127.2, 128.2, 128.5, 128.6, 133.0, 134.3,

140.3.

¹H NMR (CDCl₃, 400 MHz) δ = 1.19–1.44 (m, 4H) 1.69 (td,

J = 12.93, 4.46 Hz, 3H) 1.74–1.84 (m, 2H) 1.84–2.13 (m, 4H)

2.56–2.70 (m, 2H) 2.79 (td, J = 12.75, 4.58 Hz, 1H) 7.16–7.26 (m, 3H)

7.26–7.34 (m, 2H)

IR ν_max(KBr): 3421 (br), 2933, 2861, 1488, 1454, 1369, 1087,

1068, 958, 884, 846, 819, 735, 699, 587 cm⁻¹

.

¹³C NMR: (CDCl₃, 100 MHz) δ = 23.0, 24.9, 29.0, 32.8, 33.2, 34.8,

59.5, 74.0, 125.8, 128.5, 128.5, 143.2.

HRMS (ESI) m/z: calculated for C₂₁H₂₃ClNO [M + H]⁺340.1663,

found 340.1662.

4.2.4. N-[(1SR,2RS)-2-Hydroxy-2-(2-phenylethyl)cyclohexyl]benzamide

(28, BB 0310746)

4.2.1.9. (1RS,2SR)-2-(Benzylamino)-1-[2-(m-tolyl)ethynyl]cyclohexanol

(24, BB 0323011). Compound 24 was synthesized according to the

general procedure GP-I from 1-[2-(m-tolyl)ethynyl]-7-oxabicyclo

[4.1.0]heptane (15) (3.58 g, 16.8 mmol, 1 eq) and benzylamine

(3.68 mL, 33.7 mmol, 2 eq) in MeCN (17 mL, 1 M), stirring the

reaction mixture at 60 °C for 12 h and isolated after ﬂash

chromatographic puriﬁcation (hexane - EtOAc, from 10:1 to 5:1) in

the amount of 1.89 g (35%) as a white solid.

To a solution of compound 27 (0.73 g, 3.3 mmol, 1 eq) in DCM

(33 mL, 0.1 M) triethylamine (0.51 mL, 3.6 mmol, 1.1 eq) was added

followed by dropwise addition of benzoyl chloride (0.43 mL, 3.6 mmol,

1 eq) and reaction mixture was stirred for 2 h. Then, mixture was

poured into water (50 mL) and extracted with DCM (3 × 50 mL). The

combined organic layers were dried over sodium sulfate and con-

centrated under reduced pressure. Crude product was puriﬁed by ﬂash

column chromatography with Hex:EtOAc from 5:1 to 1:1 as eluent to

aﬀord 0.91 g of the desired product.

R_f= 0.40 (Hexane:EtOAc, 5:1).

¹H NMR: (CDCl₃, 400 MHz) δ = 1.27–1.42 (m, 2H), 1.49–1.58 (m,

1H), 1.68–1.83 (m, 3H), 2.18–2.24 (m, 2H), 2.32 (s, 3H), 2.44–2.48

(dd, J = 11.0 Hz, 3.6 Hz, 1H), 3.75 (d, J = 13.1 Hz, 1H), 4.05 (d,

J = 13.1 Hz, 1H), 4.44 (br. s., 1H), 7.11–7.13 (m, 1H) 7.17–7.29 (m,

4H), 7.33–7.39 (m, 4H).

R_f= 0.50 (Hex:EtOAc, 1:1). Yield 85%.

¹H NMR: (DMSO‑d₆, 400 MHz) δ = 1.26–1.52 (m, 4H), 1.68–1.97

(m, 5H), 2.12–2.17 (m, 1H), 2.59–2.65 (dd, J = 11.4, 3.9 Hz, 1H),

2.83–2.89 (m, 1H), 3.66 (br. s, 1H), 4.08–4.12 (m, 1H), 6.05 (d,

J = 12.8 Hz, 1H), 7.16–7.29 (m, 5H), 7.42–7.54 (m, 3H), 7.75–7.77 (m,

2H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 21.0, 23.3, 25.1, 28.9, 38.2, 50.6,

65.0, 72.2, 86.3, 90.0, 122.6, 127.0, 128.0, 128.4, 128.6, 129.1, 131.8,

138.1, 139.9, 149.6.

¹³C NMR: (DMSO‑d₆, 100 MHz) δ = 23.8, 25.6, 28.4, 29.9, 34.5,

37.5, 52.0, 73.3, 125.7, 127.2, 128.4, 128.5, 128.7, 132.0, 133.7,

143.2, 169.31.

IR ν_max(KBr): 3458 (br.), 2933, 2858, 1452, 1070, 908, 785, 733,

692 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₂₂H₂₆NO [M + H]⁺320.2009,

IR ν_max(KBr): 3275 (br.), 2931, 2860, 1626, 1576, 1537, 1489,

found 320.2012.

1456, 1340, 1317, 1290, 698 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₂₁H₃₀NO [M + H]⁺324.1958,

4.2.2. (1RS,2SR)-2-(Benzylamino)-1-(2-phenylethyl)cyclohexanol

BB 0310495)

(25,

found 324.1950.

2-(Benzylamino)-1-(phenylethynyl)cyclohexanol²⁹(1.00 g, 3.27 mmol,

1 eq) was dissolved in methanol (10 mL) followed by addition of 10% Pd/

C (0.1 g). The resulting mixture was vigorously stirred at hydrogen at-

mosphere (1 barr) and room temperature for 18 h. After this, mixture was

ﬁltered through a thin layer of celite and solvent was removed under re-

duced pressure to give 0.78 g of desired product.

4.2.5. N-[(1SR,2RS)-2-Hydroxy-2-(2-phenylethyl)cyclohexyl]

benzenesulfonamide (29, BB 0310747)

To a solution of compound 27 (0.77 g, 3.5 mmol, 1 eq) in DCM

(35 mL, 0.1 M) triethylamine (0.54 mL, 3.8 mmol, 1.1 eq) was added

followed by dropwise addition of benzenesulfonyl chloride (0.49 mL,

3.8 mmol, 1.1 eq). The resulting mixture was stirred for 2 h and then

poured into water (50 mL). The mixture was extracted with DCM

(3 × 50 mL), combined organic layers were dried over anhydrous so-

dium sulfate and concentrated under reduced pressure Crude product

was puriﬁed by ﬂash column chromatography with Hex:EtOAc from 5:1

to 1:1 as eluent to aﬀord 0.99 g of the desired product.

R_f= 0.40 (Hex:EtOAc, 5:1). Yield 78%.

¹H NMR: (CDCl₃, 400 MHz) δ = 1.18–1.37 (m, 5H), 1.60–1.97 (m,

4H), 2.04–2.09 (m, 2H), 2.48–2.51 (dd, J = 11.4, 3.9 Hz, 1H),

2.57–2.65 (m, 1H), 2.79–2.86 (m, 1H), 3.19 (br. s, 1H), 3.66 (d,

J = 12.8 Hz, 1H), 3.93 (d, J = 12.8 Hz, 1H), 7.14–7.17 (m, 1H),

7.22–7.29 (m, 5H), 7.34–7.36 (m, 4H).

R_f= 0.60 (Hex:EtOAc, 1:1). Yield 78%.

¹³C NMR: (CDCl₃, 100 MHz) δ = 22.8, 25.2, 28.0, 29.0, 34.1, 34.5,

52.0, 65.8, 73.3, 125.7, 127.2, 128.3, 128.4, 128.5, 142.2.

IR ν_max(KBr): 3236 (br.), 3022, 2962, 2931, 2862, 2850, 1495,

¹H NMR: (DMSO‑d₆, 400 MHz) δ = 1.17–1.31 (m, 4H), 1.44–1.62

(m, 3H), 1.66–1.84 (m, 2H), 1.95–1.99 (m, 1H), 2.30 (br. s, 1H),

2.51–2.59 (td, J = 12.9, 5.3 Hz, 1H), 2.76–2.83 (td, J = 12.8, 4.6 Hz,

1H), 3.09–3.14 (m, 1H), 4.82 (d, J = 8.3 Hz, 1H), 7.16–7.29 (m, 5H),

7.47–7.60 (m, 3H), 7.89–7.91 (m, 2H).

1452, 1151, 1051, 735, 700 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₂₁H₂₈NO [M + H]⁺310.2165,

found 310.2171.

¹³C NMR: (DMSO‑d₆, 100 MHz) δ = 20.9, 21.3, 27.2, 28.4, 33.1,

58.0, 71.6, 125.8, 126.7, 128.7, 128.7, 129.5, 132.5, 142.9, 143.4.

IR ν_max(KBr): 3479, 3205, 2933, 2868, 1448, 1333, 1313, 1182,

4.2.3. (1RS,2SR)-2-Amino-1-(2-phenylethyl)cyclohexanol

0323267)

(27,

BB

Compound 25 (1.00 g, 4.6 mmol, 1 eq) was dissolved in methanol

(10 mL) followed by addition of 10% Pd/C (0.1 g). The resulting mix-

ture was vigorously stirred at hydrogen atmosphere (1 barr) and room

1157, 1072, 1039, 924, 752, 727, 692, 604, 565 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₂₀H₂₄NO₃S [M−H]⁻358.1482,

found 358.1484.

8

A. Altieri, et al.

Bioorganic&MedicinalChemistry28(2020)115717

4.2.6. But-3-en-1-yn-1-ylbenzene (31)

2H), 2.20–2.30 (m, 2H), 2.41–2.49 (m, 2H), 6.42 (t, J = 6.7 Hz, 1H),

Compound 31 was obtained according the literature procedure.³⁰

The crude product was aﬀorded after evaporation of the solvent in

vacuo and was puriﬁed by ﬂash column chromatography with n-pen-

tane as eluent to aﬀord 1.8 g of the desired product as colorless oil.

Yield 92%.

7.26–7.34 (m, 3H), 7.43 (dd, J = 7.8, 1.7 Hz, 2H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 26.7, 26.7, 29.4, 32.3, 34.4, 86.9,

93.1, 124.0, 127.1, 127.7, 128.3, 131.4, 140.4.

¹H NMR: (CDCl₃, 400 MHz) δ = 5.56 (dd, J = 11.1, 2.1 Hz, 1H),

5.75 (dd, J = 17.6, 2.1 Hz, 1H), 6.04 (dd, J = 17.6, 11.2 Hz, 1H),

7.29–7.36 (m, 3H), 7.43–7.50 (m, 2H).

4.2.11. 1-(2-Phenylethynyl)-8-oxabicyclo[5.1.0]octane (36, BB 0323269)

To

a solution of 1-(2-phenylethynyl)cycloheptene (35) (8 g,

40 mmol, 1 eq) in DCM (100 mL) at 0 °C m-CPBA (70%) (11.03 g,

61 mmol, 1.5 eq) was added portionwise. After 4 h the rsulting mixture

was poured into aqueous solution of NaOH (5%) and extracted with

DCM (3 × 100 mL). Combined organic layers were dried over anhy-

drous sodium sulfate and concentrated under reduce pressure. Crude

mixture was passed through a thin layer of SiO₂and washed with

Hex:EtOAc 3:1 system (2 × 100 mL). Solvent was removed in vacuo to

obtain 8.27 g of desired product. Yield 95%.

4.2.7. 2-(Phenylethynyl)oxirane (32)

Compound 32 was obtained according the literature procedure.³¹

Flash chromatographic puriﬁcation (hexane – EtOAc, 50:1) aﬀorded 32

in the amount of 1.86 g as a yellow oil. Yield 96%.

¹H NMR: (CDCl₃, 400 MHz) δ = 3.02 (d, J = 3.3 Hz, 2H), 3.60 (t,

J = 3.4 Hz, 1H), 7.31–7.36 (m, 3H), 7.44–7.48 (m, 2H).

¹H NMR: (CDCl₃, 400 MHz) δ = 1.35–1.47 (m, 1H), 1.51–1.70 (m,

5H), 1.78–1.89 (m, 1H), 2.03–2.14 (m, 2H), 2.23–2.32 (m, 1H), 3.40

(dd, J = 6.9, 3.5 Hz, 1H), 7.28–7.33 (m, 3H), 7.42–7.46 (m, 2H).

4.2.8. 1-(Benzylamino)-4-phenyl-but-3-yn-2-ol (33, BB 0305187)

To a solution of 2-(phenylethynyl) oxirane (32) (1.23 g, 8.5 mmol,

1 eq) in 5 mL of water benzylamine (1.12 mL, 17 mmol, 2 eq) was added

and reaction mixture was vigorous stirred for 19 h. After completion of

the reaction, mixture was poured into water (50 mL) and extracted with

Et₂O (3x50 mL). Combined organic layers were dried over anhydrous

Na₂SO₄and solvent was removed under reduced pressure. Crude pro-

duct was puriﬁed by ﬂash column chromatography with Hex:EtOAc

from 20:1 to 10:1 as eluent to aﬀord 0.4748 g of the desired product.

4.2.12. (1RS,2SR)-2-(Benzylamino)-1-(2-phenylethynyl)cycloheptanol

hydrobromide (37, BB 0310748)

A mixture of 1-(phenylethynyl)-8-oxabicyclo[5.1.0]octane (36)

(4.77 g, 22.4 mmol, 1 eq) and benzylamine (13 mL, 119 mmol, 5 eq)

was heated to 100 °C under stirring for 2 days. Then reaction mixture

was poured into water (100 mL) and extracted with CH₂Cl₂(3x100 mL).

Combined organic layers were dried over anhydrous sodium sulfate and

concentrated. Crude product was puriﬁed by ﬂash column chromato-

graphy with Hex:EtOAc from 10:1 to 3:1 as eluent.

R_f= 0.50 (hexane – EtOAc, 10:1). Yield 22%.

¹H NMR (CDCl₃, 400 MHz) δ = 2.65 (br. s., 2H) 3.00 (d,

J = 5.50 Hz, 2H) 3.93 (s, 2H) 4.70 (t, J = 5.50 Hz, 1H) 7.28–7.39 (m,

8H) 7.40–7.46 (m, 2H).

R_f= 0.60 (Hex:EtOAc, 5:1). Yield 21%.

¹³C NMR: (CDCl₃, 100 MHz) δ = 53.4, 54.3, 61.1, 85.0, 88.7, 122.5,

127.3, 128.2, 128.6, 131.8, 131.8, 139.5.

To obtain a sample of analytical purity in addition to the ﬂash

chromatography compound 37 (1 g) was dissolved in 1 mL of absolute

ethanol, ethanolic hydrogen bromide (3.3 mL, 1 equiv, 1 M solution in

abs. ethanol) was added followed by diethyl ether (1 mL). The resulting

white solid was ﬁltered oﬀ, washed with small portions of diethyl ether

and dried on air to aﬀord a white solid of analytical purity with

m.p. = 194–195 °C.

IR ν_max(KBr): 2925, 1599, 1491, 1455, 1318, 1101, 1060, 876, 758,

742, 699 cm⁻¹

.

HRMS (ESI) m/z: calculated for C₁₇H₁₈NO [M + H]⁺252.1383,

found 252.1379.

¹H NMR: (DMSO‑d₆, 400 MHz) δ = 1.19–1.39 (m, 1H), 1.45–1.82

(m, 6H), 1.96–2.09 (m, 2H), 2.76 (t, J = 8.7 Hz, 1H), 4.28 (br. s., 2H),

6.56 (s, 1H), 7.41–7.47 (m, 6H), 7.50–7.56 (m, 2H), 7.58 (dd, J = 7.3,

1.7 Hz, 2H), 8.44 (br. s., 1H), 9.01 (br. s., 1H).

4.2.9. 1-(2-Phenylethynyl)cycloheptanol (35, BB 0305189)

To a solution of phenylacetylene (30) (21.5 mL, 0.19 mol, 1 eq) in

THF (200 mL) at −78 °C and inert atmosphere 2.5 M solution of nBuLi

in hexane (86 mL, 0.21 mol, 1.1 eq) was added dropwise. After 5 min of

stirring, solution of cycloheptanone (23 mL, 0.19 mol, 1 eq) in THF

(200 mL) was added dropwise. The resulting solution was allowed to

warm up to rt, poured into water (750 mL) and extracted with DCM

(3 × 200 mL). The combined organic layers were dried over anhydrous

sodium sulfate and concentrated under reduced pressure to obtain

37.82 g of desired product. Yield 91%.

¹³C NMR: (DMSO‑d₆, 100 MHz) δ = 20.7, 23.1, 24.7, 25.5, 40.4,

48.1, 64.9, 71.5, 86.8, 88.3, 121.7, 128.7, 128.8, 129.1, 129.1, 130.4,

131.2, 131.7

IR ν_max(KBr): 3257, 2937, 2862, 2817, 1560, 1454, 1425, 1402,

1392, 1041, 1028, 750, 688 cm⁻¹

.

HRMS (ESI) m/z: Calc. for C22H26NO [M + H]⁺320.2009, found

¹H NMR: (CDCl₃, 400 MHz) δ = 1.54–1.78 (m, 6H), 1.85 (dt,

J = 6.6, 3.2 Hz, 1H), 1.88–1.99 (m, 2H), 2.05–2.20 (m, 3H), 3.69–3.81

(m, 1H), 7.27–7.33 (m, 3H), 7.38–7.47 (m, 2H).

320.2011.

4.2.13. 4-(2-Phenylethynyl)tetrahydropyran-4-ol (38, BB 0263234)

Compound 38 was obtained according the literature procedure.³²

Flash chromatographic puriﬁcation (hexane-DCM-EtOAc gradient from

2:1:0.3 to 2:1:1.5) aﬀorded 38 in the amount of 20.86 g as a white solid

of with m.p. = 54–56 °C. Yield 90%.

4.2.10. 1-(2-Phenylethynyl)cycloheptene (35, BB 0305186)

To a solution of 1-(2-phenylethynyl)cycloheptanol (34) (37.82 g,

0.17 mol, 1 eq) in 1,2-dichloroethane (176 mL, 1 M) Et₃N (98 mL,

0.53 mol, 3 eq) was added followed by dropwise addition of methane-

sulfonyl chloride (21 mL, 0.25 mol, 1.5 eq) and reaction mixture was

heated under reﬂux for 12 h. Then, the resulting mixture was allowed to

cool down to rt, poured into water (600 mL) and extracted with 1,2-

dichloroethane (2 × 150 mL). The combined organic layers were dried

over anhydrous sodium sulfate and concentrated under reduce pres-

sure. Distillation under reduce pressure of crude mixture was able to

obtain 16.66 g of desired product (b.p. = 150–170 °C, 10 mm Hg). Yield

49%.

¹H NMR (CDCl₃, 400 MHz), δ = 1.91 (ddd, J = 12.9, 9.3, 4.0 Hz,

2H), 2.00–2.09 (m, 2H), 3.74 (ddd, J = 11.9, 9.2, 2.8 Hz, 2H), 3.97 (dt,

J = 11.9, 4.5 Hz, 2H), 7.29–7.37 (m, 3H), 7.41–7.48 (m, 2H).

¹³C NMR (CDCl₃, 100 MHz), δ = 40.0 (2C), 64.9 (2C), 66.2, 85.1,

91.2, 122.3, 128.3 (2C), 128.5, 131.6 (2C).

IR υ_max, (KBr): 3356, 2962, 2933, 2870, 2218 (w.), 1338, 1290,

1230, 1153, 1095, 1084, 987, 842, 760, 696 cm⁻¹

.

HRMS (ESI): Calc. for C₁₃H₁₅O₂[M + H]⁺203.1067, found

+

¹H NMR: (CDCl₃, 400 MHz) δ = 1.51–1.68 (m, 4H), 1.73–1.83 (m,

203.1054.

9

A. Altieri, et al.

Bioorganic&MedicinalChemistry28(2020)115717

4.2.14. tert-Butyl 4-hydroxy-4-(2-phenylethynyl)piperidine-1-carboxylate

(39, BB 0263264)

1240, 1169, 957, 912, 756, 733, 690 cm⁻¹

.

HRMS (ESI): Calc. for C₁₈H₂₂NO₂[M + H]⁺284.1645, found

Compound 39 was obtained according the literature procedure.³³

Flash chromatographic puriﬁcation (hexane-DCM-EtOAc gradient from

4:1:0.5 to 4:1:2.5) aﬀorded 39 in the amount of 30.62 g as colorless oil.

Yield 70%.

284.1649.

4.2.17. 6-(2-Phenylethynyl)-3,7-dioxabicyclo[4.1.0]heptane (42, BB

0263236)

To a solution of 40 (9.2 g, 50 mmol) in benzene (500 mL) Na₂HPO₄

(42 g, 296 mmol, 6 eq) and 70% m-CPBA (15 g, ∼ 60 mmol, 1.2 eq)

were added and the mixture was vigorously stirred for 1 h. Then ad-

ditional portion of 70% m-CPBA (6 g, ∼25 mmol, 0.5 eq) was added

and the reaction was stirred for an additional 2 h and left overnight. The

mixture was then thoroughly washed twice with a solution of K₂CO₃

(30 g) and Na₂SO₃(15 g) in water (500 mL), the organic layer was se-

parated and the aqueous was combined and extracted with hexane-

DCM 4: 1 mixture (250 mL). The combined organic extracts were dried

with Na₂SO₄, the solvents were evaporated in vacuo to give the crude of

42. The residue was puriﬁed by using chromatography (SiO₂, hexane-

DCM-EtOAc 10:1:1.5) to aﬀord 42 in the amount 4.46 g as colorless oil.

Yield 45%.

R_f= 0.50 (Hexane:EtOAc, 2:1).

¹H NMR (CDCl3, 400 MHz), δ = 1.47 (s, 9H), 1.74–1.85 (m, 2H),

1.95–2.05 (m, 2H), 2.39 (br. s., 1H), 3.33 (ddd, J = 13.4, 9.7, 3.3 Hz,

2H), 3.84 (br. s., 2H), 7.28–7.37 (m, 3H), 7.40–7.48 (m, 2H).

¹³C NMR (CDCl₃, 100 MHz), δ = 28.4 (3C), 39.1 (2C), 40.7 (br.),

41.2 (br.), 67.2, 79.7, 85.3, 91.0, 122.2, 128.3 (2C), 128.6, 131.7 (2C),

154.7.

IR υ_max, (KBr): 3207 (br.), 2960, 2931, 2856, 2216 (w.), 1697, 1421,

1365, 1277, 1248, 1173, 1144, 1068, 754, 690 cm⁻¹

.

HRMS (ESI): Calc. for C18H23NO3Na⁺[M + Na]⁺324.1570,

found 324.1570.

4.2.15. 4-(2-Phenylethynyl)-3,6-dihydro-2H-pyran (40, BB 0263235)

To a solution of compound 38 (0.10 mol.) was added dropwise over

5 min MsCl (14 g, 0.12 mol, 1.2 eq). The mixture was stirred for 30 min

at −30 °C, the cooling bath was removed and the reaction was stirred at

room temperature for an additional 24 h. The mixture was then treated

with 5% H₂SO₄(350 mL), the organic layer was separated and the

aqueous was extracted with a hexane-DCM 10: 1 mixture (200 mL).

Organic extracts were combined, dried with Na₂SO₄, the solvents were

evaporated in vacuo. The residue was puriﬁed by using chromato-

graphy (SiO2, hexane-DCM-EtOAc 10: 1: 1) to aﬀord 40 in the amount

17.1 g as colorless oil. Yield 87%

R_f= 0.40 (Hex:EtOAc 4: 1).

¹H NMR (CDCl₃, 400 MHz), δ = 2.20–2.36 (m, 2H), 3.50 (d,

J = 2.5 Hz, 1H), 3.54 (d, J = 5.1 Hz, 1H), 3.55 (d, J = 5.0 Hz, 1H), 3.96

(d, J = 13.5 Hz, 1H), 4.11 (dd, J = 13.5, 2.9 Hz, 1H), 7.28–7.38 (m,

3H), 7.41–7.49 (m, 2H).

¹³C NMR (CDCl3, 100 MHz), δ = 39.9, 48.7, 58.2, 61.1, 64.4, 83.2,

87.4, 121.8, 128.3 (2C), 128.8, 131.8 (2C).

IR υ_max, (KBr): 2958, 2850, 2231 (w.), 1491, 1298, 1242, 1128, 870,

758, 733, 692 cm⁻¹

.

HRMS (ESI): Calc. for C₁₃H₁₃O₂[M + H]⁺210.0910, found

210.0909.

R_f= 0.60 (Hex:EtOAc 4: 1).

4.2.18. tert-Butyl 6-(2-phenylethynyl)-7-oxa-3-azabicyclo[4.1.0]heptane-

3-carboxylate (43, BB 0263266)

¹H NMR (CDCl₃, 400 MHz), δ = 2.32–2.39 (m, 2H), 3.84 (t,

J = 5.3 Hz, 2H), 4.26 (q, J = 2.8 Hz, 2H), 6.15–6.20 (m, 1H), 7.28–7.36

(m, 3H), 7.41–7.48 (m, 2H).

To a solution of 41 (14 g, 49.5 mmol) in benzene (500 mL) Na₂HPO₄