Multicomponent, fragment-based synthesis of polyphenol-containing peptidomimetics and their inhibiting activity on beta-amyloid oligomerization

View Article Online

View Journal

Organic &

Biomolecular

Chemistry

Accepted Manuscript

This article can be cited before page numbers have been issued, to do this please use: C. Lambruschini,

D. Galante, L. Moni, F. Ferraro, G. gancia, R. Riva, A. Traverso, L. Banfi and C. D'Arrigo, Org. Biomol.

Chem., 2017, DOI: 10.1039/C7OB02182H.

This is an Accepted Manuscript, which has been through the

Volume 14 Number

1 7 January 2016 Pages 1–372

Royal Society of Chemistry peer review process and has been

accepted for publication.

Organic &

Biomolecular

Chemistry

www.rsc.org/obc

Accepted Manuscripts are published online shortly after

acceptance, before technical editing, formatting and proof reading.

Using this free service, authors can make their results available

to the community, in citable form, before we publish the edited

article. We will replace this Accepted Manuscript with the edited

and formatted Advance Article as soon as it is available.

You can find more information about Accepted Manuscripts in the

author guidelines .

Please note that technical editing may introduce minor changes

to the text and/or graphics, which may alter content. The journal’s

standard T erms & Conditions and the ethical guidelines, outlined

in our author and reviewer resource centre, still apply. In no

event shall the Royal Society of Chemistry be held responsible

for any errors or omissions in this Accepted Manuscript or any

consequences arising from the use of any information it contains.

ISSN 1477-0520

COMMUNICATION

Takeharu Haino et al.

Solvent-induced emission of organogels based on tris(phenylisoxazolyl)

benzene

rsc.li/obc

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 1 of 20

View Article Online

DOI: 10.1039/C7OB02182H

Organic and Biomolecular Chemistry

ARTICLE

Multicomponent, Fragment-Based, Synthesis of Polyphenol-

containing Peptidomimetics and their Inhibiting Activity on Beta-

Amyloid Oligomerization

Received 00th January 20xx,

Accepted 00th January 20xx

Chiara Lambruschini,^a† Denise Galante,^b† Lisa Moni,^a† Francesco Ferraro,^aGiulio Gancia,^bRenata

Riva,^aAlessia Traverso,^aLuca Banfi^a* and Cristina D'Arrigo^b*

DOI: 10.1039/x0xx00000x

www.rsc.org/

A new and concise fragment-based approach towards artificial (but "natural-based") complex polyphenols has been

developed, exploiting the Ugi multicomponent reaction of phenol-containing simple substrates. The resulting library of

compounds has been tested for the capacity to inhibit β-amyloid protein aggregation, as a possible strategy to develop new

chemical entities to be used as a prevention or a therapy for Alzheimer's disease. Some of the members of the library have

demonstrated, in Thioflavin assays, a highly promising activity in inhibiting aggregation for two β-amyloid peptides: Aβ1-42

and the truncated AβpE3-42.

type ring, making them highly susceptible to oxidation, thus

14

strongly reducing their half-life in the body.^12,

Finally,

Introduction

chemical modifications of complex natural polyphenols is quite

tricky,¹⁵hampering the systematic synthesis of analogues that

might overcome the above quoted limitations and/or be

endowed of higher potency.

Alzheimer’s Disease (AD) is the most prevalent

neurodegenerative disorder. The hallmarks of AD are the

extracellular plaques, derived from aggregation of β-amyloid

peptides, and neurofibrillary tangles composed by

hyperphosphorylated protein tau. Inhibition of β-amyloid

protein aggregation represents one of the most promising

targets in the development of pharmacological treatments for

the prevention of Alzheimer's disease.^1-3Moreover, substances

that strongly bind to β-amyloid proteins may be very useful

diagnostic tools for an early detection of this disease.⁴Among

the various substances that have been found to bind to β-

amyloids, natural polyphenols have emerged as a particularly

promising class, being able to inhibit β-amyloid aggregation and

disrupt preformed amyloid fibrils.^5-11Hydrogen bonding,

hydrophobic interactions, and aromatic stacking are suggested

to be the driving forces of the anti-amyloidogenic role of

polyphenols. In addition, antioxidant activity may also be

involved in the anti-amyloidogenic role.¹²Figure 1 depicts some

of the most active natural polyphenols.

However, these natural compounds have often poor

pharmacokinetic properties. For example, pharmacokinetic

results for curcumin and its metabolites suggested limited or

very poor bioavailability; in particular, curcumin was present in

very little amount in the cerebrospinal fluid.¹³In addition,

several natural polyphenols contain a catechol or a pyrogallol

^a.Department of Chemistry and Industrial Chemistry, University of Genova, via

Dodecaneso 31 - 16146 Genova, Italy. E-mail: banfi@chimica.unige.it.

^b.Istituto per lo Studio delle Macromolecole, Consiglio Nazionale delle Ricerche, via

De Marini 6, 16149 Genova, Italy. E-mail: cristina.darrigo@ge.ismac.cnr.it.

^c.Address here.

Figure 1 Some natural polyphenols with β-amyloid anti-aggregation properties.

† Contributed equally to this work

Electronic Supplementary Information (ESI) available: [details of any supplementary

information available should be included here]. See DOI: 10.1039/x0xx00000x

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 1

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 2 of 20

ARTICLE

Organic and Biomolecular Chemistry

Therefore, we reasoned that a fragment-based synthesis of

natural-derived polyphenols, obtained by joining simple,

monocyclic, phenol containing, building blocks, would be a very

useful tool to assembly a huge number of molecular entities,

allowing: a) optimization of pharmacodynamic properties; b)

optimization of pharmacokinetic properties; c) the synthesis of

structures that include pharmacophores directed towards

alternative AD-related targets, with the aim to develop drugs

able to simultaneously interact with different targets (multi-

target strategy).¹⁵If the synthetic sequence is smartly designed,

in order to be quite short, and simple building blocks derived

from renewable sources are exploited, the final optimized

compounds could be easily accessible in an eco-friendly

manner, thus making their potential use as nutraceutics

definitely feasible.

View Article Online

DOI: 10.1039/C7OB02182H

Multicomponent reactions have emerged in the last 20 years as

a powerful tool in drug discovery. They are intrinsically

endowed with very high step economy and operational

simplicity and are thus perfectly suited for a rapid generation of

libraries characterized by several diversity inputs. Among them,

the isocyanide-based Ugi reaction (U-MCR)¹⁶is particularly

useful, since it allows the simultaneous joining of 4 diversity

inputs, represented by easily accessible compounds, as

isocyanides, aldehydes, primary amines and carboxylic acids.

Thus, also taking advantage of our previous experience both in

the Ugi reaction,^17-20and in the assembly of natural-based

polyphenols,²¹we selected U-MCR at the key step in our

fragment-based approach. The classical scaffold obtained by

the Ugi reaction is a peptidomimetic structure. This is another

added value of our approach, since also peptidomimetics are

widely studied as potential inhibitors of β-amyloid

aggregation.²²

Using this synthetic methodology we were able to prepare a

series of complex polyphenols containing 2 to 4 hydroxy-

substituted aryl groups, most of them derived from renewable

sources, and test them for their ability to inhibit in vitro β-

amyloid aggregation. In this paper we report our preliminary

observations, representing a first "proof of concept" for our

approach.

Scheme 1 General synthetic strategy

and the easiness of final deblocking, we opted for a simple

acetyl group.²¹As shown in Scheme 2, a first Ugi reaction with

only two phenol containing components (aniline 3^23,and

aldehyde 4²⁵) worked well, affording compound 2a in good

yields. It was then smoothly deprotected by saponification to

the diphenol 1a. However, the use of the acetyl as protecting

group was soon demonstrated to be far from general. For

example, simply using the protected phenol containing

24

isocyanide

Similarly, all attempted Ugi reaction using diacetyl protected

caffeic acid were unsuccessful. We think that the aryl acetates

5, we failed to isolate any of the expected product 6.

7

are somehow unstable under the Ugi reaction and that the

liberated phenols and acetic acid promote unwanted side

reactions. Thus, only with protected hydroxyanilines and

reactive isocyanide/carboxylic acids the reaction turned out to

be feasible, strongly limiting diversity exploration.

Looking for a more stable protection we shifted to the dimethyl-

tert-butylsilyl group (TBDMS). In this case the group proved to

be fully stable under the Ugi conditions. However, the reactions

tend to be rather sluggish. An improvement can be obtained by

preforming the imine treating the aldehyde and the amine in

CH₂Cl₂in the presence of dry MgSO₄. Anyway, the isolated yield,

in the case of compound 11, was only 25%. Furthermore, the

presence of more than two TBDMS groups renders the Ugi

products rather insoluble in most solvents making their

purification, as well as the assessment of purity by NMR or

HPLC, troublesome. In particular, at NMR, broad signals due to

slowly converting conformers are observed. Therefore, because

of the poor atom economy, the slow reaction kinetics and the

unsatisfactory chemico-physical properties, we decided to

abandon this protecting group as well.

Our attention was then drawn by the allyl group for its high

atom economy (similar to the acetyl), its expected stability

under the Ugi conditions, and the possibility to remove it under

neutral conditions, thanks to palladium (0) catalysis. The Ugi

reaction of substrates containing this group was thoroughly

optimized. Scheme 3 shows a representative example. In

particular we noticed that benzaldehydes bearing an allyloxy

Results and discussion

Synthesis

As depicted in Scheme 1, the Ugi reaction allows us to prepare

a peptidomimetic structure

1 with 4 appendages. In our plan,

from two to four of these appendages should contain a phenolic

aryl group, tethered to the main scaffold through linkers of

different lengths. In principle, our target compounds 1 could be

accessed in just one step by employing, in the U-MCR,

components containing the free phenols. However, preliminary

investigation has shown that the presence of free phenols had

a negative effect on the yield and cleanliness of the

multicomponent reaction. Thus we shifted to a slightly longer (2

steps) sequence, employing suitably protected building blocks.

Many efforts were devoted to the selection of the best

protecting group. Initially, because of the high atom economy

2 | J. Name., 2012, 00, 1-3

This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

Page 3 of 20

Organic & Biomolecular Chemistry

Please do not adjust margins

Organic and Biomolecular Chemistry

ARTICLE

View Article Online

DOI: 10.1039/C7OB02182H

Scheme 2 Synthesis of the first polyphenols using the Ac or TBDMS protecting groups.

group in para position resulted less reactive (because of the biophysical tests, by basic solvolysis, acid resin treatment, and

electron-donating properties of allyloxy) than 4- filtration, avoiding extractive or chromatographic treatments,

acetoxybenzaldehyde

4 or benzaldehyde and that substituted that may be troublesome, because of the polyphenol polarity

cinnamic acids such as protected ferulic and caffeic acids also and/or for the possible partial degradation. This procedure

brought about a slower kinetic. We found that the best solvent, (procedure B) is exemplified in Scheme 3 for the synthesis of

in order to have reasonable reaction times and avoid the compound 1d. Apart from some products synthesized in the

formation of Passerini side-products, was a 1:1 mixture of EtOH initial part of this research, we later routinely used procedure B.

and trifluoroethanol. Moreover, it was advantageous to In this case, full characterization was carried out only on the

preform the imine in this solvent for 5 h, before adding the acetylated compounds

other reagents. The reactions typically last 48-72 h.

2

. The final phenols derived from

1

deacetylation were pure enough for testing, as checked by H

As for the deblocking step, we tried various Pd catalysts and NMR and HPLC (HPLC purity ≥ 96% in nearly all cases, except for

scavengers. Eventually, the reaction was found to be more 1d 1l and 1m (92%)). Procedure B is also useful in ensuring the

,

reproducible using a palladium (II) precursor, Pd(PPh₃)₂Cl₂, than complete removal of palladium from our polyphenols. Also

with Pd(PPh₃)₄. As scavenger, ammonium formate was the best, when using method A, the residual content of palladium was,

allowing an easy removal of its excess by a simple extraction after chromatography, negligible. To prove this, in the case of

under neutral conditions. In the case of compound 1c, which 1c we have tested two different samples. The first one was

was initially used as model, chromatographic purification, simply chromatographed after allyl deblocking. The second one

followed by treatment with active coal, worked fine, affording a was treated, before chromatography, with a known palladium

very pure product (procedure A). However, in other cases, scavenger, that is Argoresin-MP-TMT. The two samples had the

especially when a catechol or a pyrogallol moiety were present, same colour and gave identical results in the biophysical tests.

direct chromatographic purification of the final polyphenol was Scheme 4 depicts all the polyphenols 1a-q prepared so far,

not fully satisfactory, due to partial degradation. Moreover, we whereas Table 1 reports the procedure used in each case and

lost some material during the work-up of the deprotection step. the yields of various steps (except for compounds 1a,b prepared

To avoid these problems, we decided to react the crude as described in Scheme 2).

polyphenols with acetic anhydride and purify, store and fully Concerning the Ugi reactions, we found out that its efficiency

characterize them as the peracetylated derivatives

2. Removal depends on the nature of components used. For example, it

of the acetyl groups was then carried out only just before worked poorer using substituted anilines than with

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 3

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 4 of 20

ARTICLE

Organic and Biomolecular Chemistry

View Article Online

DOI: 10.1039/C7OB02182H

Scheme 3 Representative procedures for the preparation of polyphenols 1 via allyl ethers.

benzylamines or other aliphatic amines. Also protected ferulic isocyanide and an aniline led to a poor yield in the Ugi reaction

and caffeic acids were somehow less reactive than simple acids (18%). In this case, we obtained a moderate improvement using

like propionic acid or benzoic acids. Allyl protected substituted 4-pivaloyloxybenzaldehyde (31%). We then used procedure B

benzaldehydes (p-hydroxybenzaldehyde or vanillin) were less and isolated and characterized the mixed pivaloyl-acetyl

reactive than benzaldehyde, probably because of the electron- compound 2k

.

donating properties of the allyloxy group in para position. Apart from the polyphenols listed in Scheme 4, other

Finally, aliphatic isocyanides behaved better than the aromatic compounds targeted by us were not obtained in reasonable

ones. In particular, during the synthesis of compound 1k, the yields.

combination of 4-allyloxybenzaldehyde, a bulky aromatic

Table 1 Yields of polyphenol synthesis using the allyl protecting group

Entry

1

2

3

4

5

6

7

8

Polyphenol

Yield of 14

69%

Procedure used^a

Yield of 2

Yield of 1

b

1c

1d

1e

1f

1g

1h

1i

1j

1k

1l

1m

1n

1o

1p

1q

A

B

A

B

A

B

-

80%

c

57% (91%)^c

76%

-

b

72%

73%

28%

22%

33%

17%

31%^e

75%

59%

63%

69%

71%

72%

-

59%

d

83%

59%

-

d

b

-

74%

68%

b

d

64%

69%

58%

78%

69%

74%

60%

75%

-

d

9

d

10

11

12

13

14

15

d

a

c

See Scheme 3 and Experimental part. ^bIn procedure A, 14 was directly converted to 1. In brackets the yield calculated taking into account the recovered

aldehyde. ^dIn procedure B, 2 was converted quantitatively into 1 by hydrolysis of acetates. ^eIn this case 4-pivaloyloxybenzaldehyde was used, and Ugi product

was not 14a, but 15 (Scheme 4).

4 | J. Name., 2012, 00, 1-3

This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 5 of 20

View Article Online

DOI: 10.1039/C7OB02182H

Organic and Biomolecular Chemistry

ARTICLE

Scheme 4 Polyphenols prepared and their precursors.

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 5

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 6 of 20

ARTICLE

Organic and Biomolecular Chemistry

For example, although triallylated gallic acid was a good

substrate for the Ugi reaction, all attempts to deprotect it

without extensive decomposition were unsuccessful. Similarly,

when we saturated the double bond in caffeic acid derived

View Article Online

DOI: 10.1039/C7OB02182H

polyacetate 2f

, deblocking of the acetyl group led to

decomposition of the final product as well. We attribute this

behaviour to the presence of a catechol or pyrogallol moiety,

which are prone to oxidation under basic conditions. In the case

of caffeic acid adducts, the conjugation with the unsaturated

amides makes the catechol less electron-rich and thus more

stable to oxidation, but when the double bond is hydrogenated

the catechol becomes too reactive.

Biochemical and biophysical assays

The first biophysical analysis performed on the new

polyphenols was the solubility in aqueous solution because the

working condition is Phosphate Buffer Solution (PBS) at pH 7.4

to mimic the physiological environment. All compounds showed

complete solubility at working concentration (25 µM) in PBS

containing 1% of DMSO (solubility-related data are reported in

the S.I.). This percentage of DMSO does not damage cells and

animals for future biological tests, and also does not alter the

aggregation of β-amyloids. In any case control blank

experiments with samples containing 1% DMSO in the buffer

were always performed in parallel.

To investigate their ability to inhibit the amyloid aggregation,

kinetics assays monitored by thioflavin-T were used to follow

the formation of β-sheet rich structures, visualized then by

transmission electron microscopy (TEM).^{26, 27}

Figure 2 ThT Fluorescence in percentage respect to the control sample, after 24 h of

aggregation at 37 °C, the concentration was 5 µM for β-amyloids and 25 µM for

polyphenols in PBS + 1% DMSO.

We explored the interaction of the new complex polyphenols

with two particular β-peptides, Aβ1-42 and AβpE3-42. The full-

length Aβ1-42 is one of the most abundantly identified in the

brain deposits (together with Aβ1-40 and N-terminal truncated

Aβ peptides). AβpE3-42 is a peptide N-terminal truncated at

residue 3 (Glu) and further modified by cyclization of Glu (E) to

pyroglutamic acid (pE). These structural modifications increase

AβpE3-42 aggregation propensity, its resistance to degradation

of proteases, and display an enhanced cytotoxicity in

comparison to Aβ1-42^{28, 29}as well as the ability to unfold the

full-length into toxic aggregates.³⁰

Before aggregation kinetics experiments, we used a natural

polyphenol as a positive control to compare the efficacy of the

new synthetic polyphenols. From the literature,³¹

epigallocatechin gallate (EGCG) was the most potent natural

polyphenol in inhibiting (in vitro) aggregation of β-amyloid

proteins, although it was also reported that in vivo has a poor

stability.¹²After investigating the lowest concentration at which

EGCG strongly inhibited amyloid aggregation, we chose to work

at 25 μM and to use this concentration also for our synthetic

polyphenols.

structure from α-helix (in the membrane environment) or coil

(in basic environment) to β-sheet conformation.³²Then the

aggregation proceeds forming small aggregates, called

oligomers, until reaching larger aggregates such as long fibrils,

which will later precipitate.

We used as reference Aβ1-42 and AβpE3-42 alone under the

same conditions of the experiments carried out in presence of

the new polyphenols. In Figure 2 it is reported, for all samples

(except 1a and 1b), the ThT emission value after 24 h of

aggregation at 37 °C. In the case of 1a and 1b, the first

polyphenols prepared by us, we carried out the test on Aβ1-42

alone and at higher concentrations. Only 1b showed moderate

activity and we did not repeat the experiments at 25 µM. The

relative data are reported in the S.I.

As we can see in panel A, all new polyphenols have some effect

on Aβ1-42 aggregation. 1m, 1f and especially 1j even increase

the β-sheets content of the peptide, so with these compounds

also the fibrils formation grows. On the contrary, the best new

polyphenol to inhibit the fibrillation process is 1c. The degree of

inhibition is also quite high for 1h, 1i, 1l and 1n, and moderate

We explored the interaction of the new complex polyphenols

with Aβ1-42 and AβpE3-42 to verify their ability to inhibit β-

amyloid aggregation. β-sheet content and aggregation process

at 37 °C in PBS (150 mM, pH 7.4) and 1% DMSO were followed

by fluorescence using Thioflavin T (ThT), a probe that detects

the presence of β-sheets in the sample. Indeed, the aggregation

of β-amyloids starts when they change their secondary

for 1d, 1e, 1k, 1o, 1p and 1q. However, with 1c also the kinetic

of aggregation slows down (see Figure 3).

For AβpE3-42 (Figure 2, panel B) the results were different, the

best inhibitor of the aggregation process being indeed 1f, but

also 1e, 1i and 1n showed a good effect. Moderate inhibition

was visible for 1l and 1q. In both cases, epigallocatechin gallate

(ECGC), the most active natural polyphenol, was still more

6 | J. Name., 2012, 00, 1-3

This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

Page 7 of 20

Organic & Biomolecular Chemistry

Please do not adjust margins

Organic and Biomolecular Chemistry

ARTICLE

effective in preventing amyloid aggregation at the same

concentration. It is however worth noting that the presence of

two pyrogallol moieties in ECGC make its stability under

physiological conditions troublesome, whereas we have

obtained a similar, albeit somehow lower, acitivity with much

View Article Online

DOI: 10.1039/C7OB02182H

more stable polyphenols derived from ferulic acid³³

(1c, 1i, 1n),

which are much more stable to oxidation and more promising

from the pharmacokinetic point of view. Is interesting to note

that for Aβ1-42 there are different polyphenols with a good

anti-aggregating effect (Inhibition>30%), while for AβpE3-42,

which is more prone to aggregation, more resistant to

degradation and more toxic in comparison to Aβ1-42,

polyphenols with a good anti-aggregating effect are fewer.

To better understand the different inhibition mechanism of the

most active molecules, we determined also the aggregation

kinetics curve of ThT Fluorescence emission over time for Aβ1-

42 and AβpE3-42 in presence of 1c and 1f (Figure 3). In the case

of Aβ1-42, 1c slows down the fibril growth phase and reduces

the amount of fibrils, while 1f slows down only the lag phase,

but the amount of fibrils is similar to that of Aβ1-42 alone as

reported by the plateau value.

On the contrary, in the case of AβpE3-42, 1c has almost no

effect on the aggregation inhibition, while 1f inhibits the

maximum Aβ assembly. AβpE3-42 is very fast to aggregate in

the initial stage: in fact, in our conditions, it is never possible to

see a lag phase for this peptide. The aggregation pathway is

different from that of the full-length peptide and results in the

enhancement of the seed production that speeds the

aggregation into more fragmented and less structured species.

We think that 1c and 1f act at different levels during

aggregation, inhibiting the formation of different structural

species. 1f is able to inhibit the formation of oligomers that

work as seed for the aggregation. In fact, it extends the lag

phase in Aβ1-42 but does not inhibit the fibril formation,

whereas is able to strongly reduce the AβpE3-42 aggregation.

Figure 4 Effect of addition of 1c to aggregated fibrils (Aβ1-42, monitored by ThT

Fluorescence emission).

So 1f is effective on AβpE3-42 because it inhibits the first phase

of aggregation, the one forming the oligomers.

As shown in Figure 4, 1c is even capable to disrupt fibrils, once

they have formed. So, addition of 1c after 24 h (when the

aggregation process in the absence of inhibitors is already

complete) provokes a significant decrease of ThT fluorescence

emission.

To confirm these data, we studied the morphology of Aβ

peptides aggregates in presence of those polyphenols that

behaved best from ThT test. They were observed after 24 h of

incubation at 37°C at the same ratio of the fluorescence

experiments (Figure 5). In Figure 5, panel A, the morphology of

Aβ1-42 is reported, showing typical amyloid fibrils that appear

as very entangled fibril bundles and also striated ribbons are

visible. When 1c is added to Aβ1-42 (panel B), fibrils bundles

decrease and the thinner fibres appear less entangled.

Moreover, the fibrils are somehow fragmented, due to the twist

Figure 3 Kinetics of aggregation monitored by ThT Fluorescence emission. A) Kinetics curve for Aβ1-42; B) Kinetics curve for AβpE3-42. The concentration was 5 µM for Aβ peptides

and 25 µM for polyphenols in PBS + 1% DMSO, in yellow are reported the standard error for the curves of Aβ alone.

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 7

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 8 of 20

ARTICLE

Organic and Biomolecular Chemistry

change along the fibre. When Aβ1-42 and 1f are mixed (panel applied. One of these was the creation of short synthetic

View Article Online

C), fibril bundles and little spheroidal aggregates appear even if peptides capable of binding Aβ but unab^Dle^Ot^I:o¹⁰b^.e¹⁰c³o⁹m^/Ce^7Op^Ba⁰r²t¹o⁸f^2Ha

the fibrils have smaller diameters than Aβ1-42 alone. The β-sheet structure (β-sheet breaker peptides) that destabilize

quantity of β-sheet in this sample is not different from that of the amyloidogenic Aβ conformer and hence preclude amyloid

full-length alone (as showed in the ThT assay). This alternative formation.³⁶These β-sheet breaker peptides act by the binding

morphology depends on amyloid multi-step assembly pathways of the central hydrophobic region of Aβ protein (amino acids 17-

that is altered from the slowdown of the lag phase by 1f

.

21: LVFFA). Another approach was the use of polyphenolic

Looking at panel D, the morphology of AβpE3-42 is shown with compounds as β-sheet inhibitors. The possible mechanisms by

few bundles and short fibrils. The addition of 1c (panel E), which polyphenols destabilize β-amyloid aggregation still

results in the decrease in the amount of fibrils but the remain unclear, but several mechanisms have been proposed to

morphology is very similar to that of the AβpE3-42 alone. As a date and structural similarities between various highly efficient

matter of fact this polyphenol is not very effective in inhibiting inhibitors have been identified. It has become evident that the

the assembly of the pyroglutamate β-amyloid. Finally, in the presence of phenolic rings with a few linkers and at least two

presence of 1f (panel F), in agreement with the ThT assay, the hydroxy groups could favour effective non-covalent

number of fibrils and their length strongly decrease and many interactions with the fibril β-sheet structures and interfere with

dispersed small spheroidal aggregates appears. Moreover, their elongation and/or assembly.³³Both the number of

globular aggregates attached along the fibrils are visible. Also hydroxy groups and the positioning of these groups on the

the morphology confirms the structural changes induced by the polyphenolic structure is important, however there is no clear

addition of the best new polyphenols.

understanding of the link between phenol positional

Figure 5 Morphology of the species by TEM. A) Aβ1-42 alone; B) Aβ1-42 + 1c; C) Aβ1-42 + 1f; D) AβpE3-42 alone; E) AβpE3-42 + 1c; F) AβpE3-42 + 1f

Several studies have indicated that hydrophobic forces, substitution and corresponding anti-aggregation activity.

aromatic stacking, and electrostatic interactions stabilize the Aβ Moreover, the planarity of the inhibitor is essential for

structure.³⁴It was found that short fragments of Aβ (QKLVFF) increasing surface contact with Aβ peptides.⁵

self-assemble and also bind specifically to full-length peptides, All of our polyphenols have a peptidomimetic structure, more

supporting the hypothesis that π-π interactions may play a than two aromatic rings essential for π–π stacking interactions

central role in the molecular recognition and Aβ self-assembly with hydrophobic amino acid residues of Aβ and at least two

process.³⁵In the last years, various approaches to inhibit and hydroxyl groups to form hydrogen bonds with hydrophilic

reverse misfolding and aggregation of β-amyloid have been amino acid residues of Aβ. The resonance structure of

8 | J. Name., 2012, 00, 1-3

This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

Page 9 of 20

Organic & Biomolecular Chemistry

Please do not adjust margins

Organic and Biomolecular Chemistry

ARTICLE

polyphenols provides enough planarity to penetrate the Aβ approach and on the demonstration that some of these

View Article Online

fibril hydrophobic grove, thus disturbing the fibril structure.³⁷

compounds are indeed able to inhibit or even disrupt β-amyloid

DOI: 10.1039/C7OB02182H

On the basis of the results collected till now, we can try to aggregation. In fact, we tested the anti-aggregation activity on

correlate the observed activity with the various two different β-amyloid peptides (Aβ1-42 and AβpE3-42),

pharmacophores. As far as it concerns the groups derived from normally present in AD brains, that have a different assembly

the isocyanide, it is noteworthy that the most active compounds pathway. For this reason, some polyphenols are more prone to

so far have a tert-butyl group as the isocyanide derived one, inhibit the aggregation process of Aβ1-42 than that of AβpE3-

suggesting that the presence of an aromatic ring in this position 42 and vice versa. This approach could allow the formulation of

is not essential.

mixtures of active polyphenols to inhibit simultaneously the

On the contrary, the structure of the residues derived from the aggregation of both peptides and avoid the formation of more

carboxylic acid and the amine seems more important. neurotoxic co-aggregates.

Regarding the first one, best results have been obtained with Clearly more insight into the mechanism by which our systems

cinnamic acid derivatives (caffeic and ferulic acid). For the inhibit β-amyloid protein aggregation is needed, for example by

pyroglutamate β-amyloid, we noted that polyphenols using NMR spectroscopy or computational models. However,

synthesized from caffeic acid (1e and 1f) are able to strong notwithstanding the still limited number of molecules tested,

inhibit aggregation, whereas among polyphenols derived from the results depicted in Figure 3 indicates that subtle variation in

ferulic acid, only 1i and 1n show good activity. On the other the structure of the appendages may have a strong impact on

hand, for the full-length peptide we noticed in most cases activity. Thus, the smart synthetic approach (based on Ugi

(except for 1m) a good inhibitory effect when the starting MCR), that allows to assemble these polyphenols in 2 steps, by

carboxylic acid is ferulic acid. The effect is good for 1c

and moderate for 1d 1o 1p and 1q. On the other hand, tuning of the pharmacophores in order to increase potency

polyphenols where carboxylic acid is a benzoic acid (1g 1j an and/or selectively target different sub-species of β-amyloid

, 1i, 1l, 1n varying up to 4 diversity inputs, will strongly facilitate the fine

,

1k) have no or little inhibitory effect on both peptides. A proteins. The incorporation of fragments with known anti-

propionyl group (1h) resulted in no effect on the truncated oxidant activity, such as ferulic acid, may have other kind of

peptide, but in a moderate activity on the full-length one. We beneficial effects on AD patients, as pointed out in a recent

can conclude that ferulic acid is best for Aβ1-42, whereas as paper.³⁸Compared to the most active natural compounds (e.g.

caffeic acid is the best for AβpE3-42, although, as shown by 1i epigallocatechin gallate), our systems are expected to be

and 1n, also ferulic acid derivatives may inhibit this peptide.

metabolically much more stable (especially those, like 1c, not

The nature of the group derived from the amine component in containing a catechol system), thus overcoming the main

the Ugi seems important for both peptides. Best results have drawback of some natural polyphenols and making them better

been obtained with benzylamines or anilines, whereas a drop of suited for in vivo experiments, that will soon be carried out.

activity was observed in the case of 1m, having just one more

carbon atom, or for 1d, where the benzyl/aryl group is replaced

by a simple butyl. The benzyl groups seem better than the aryl

Experimental

ones (the most promising compounds, 1c and 1f have indeed a

simple benzyl group), although it is remarkable that 4-

hydroxyphenyl containing 1i is more active than 1c for the

truncated peptide.

Finally, the group derived from the aldehyde has been so far less

explored by us. However, in particular for the pyroglutamate β-

amyloid peptide, we have noticed a remarkable influence of an

additional methoxy group (compare compound 1c, containing

the 4-hydroxyphenyl group, and 1n, containing the 4-hydroxy-

3-methoxyphenyl group).

It is interesting to note that only 1n and 1i are able to inhibit the

aggregation of both Aβ1-42 and AβpE3-42. This great variation

of substituent effects on the two β-amyloid peptides likely

depends on their intrinsic differences. Probably our polyphenols

bind in different region of the chain by interacting with diverse

residues and/or at distinct levels in the assembly mechanism

that brings to the aggregation.

NMR spectra were taken at r.t. in CDCl₃or in d₆-DMSO at 300

MHz (¹H), and 75 MHz (¹³C), using, as internal standard, TMS (¹H

NMR in CDCl₃; 0.000 ppm) or the central peak of DMSO (¹H NMR

in d₆-DMSO; 2.506 ppm) or the central peak of CDCl₃(¹³C in

CDCl₃; 77.02 ppm), or the central peak of DMSO (¹³C in d₆-

DMSO; 39.43 ppm). Chemical shifts are reported in ppm (



scale). Peak assignments were made with the aid of gCOSY and

gHSQC experiments. In ABX system, the proton A is considered

upfield and B downfield. [α]_Dvalues are given in 10⁻¹deg cm²g⁻¹.

IR spectra were recorded as solid, oil, or foamy samples, with

the ATR (attenuated total reflectance) technique. TLC analyses

were carried out on silica gel plates and viewed at UV (=254

nm or 360 nm) and developed with Hanessian stain (dipping

into a solution of (NH₄)₄MoO₄·4H₂O (21 g) and Ce(SO₄)₂·4H₂O (1

g) in H₂SO₄(31 mL) and H₂O (469 mL) and warming). R_fvalues

were measured after an elution of 7–9 cm. GC-MS analysis were

recorded on HP-5890 series II HEWLETT PACKARD equipped

with a HP-1 column (12 m, Ø = 0.2 mm) using He as carrier gas.

MS were recorded on an electronic impact (EI, 70 eV) HP-5971A

detector. Chromatography condition: flow 1.0 mL/min, injector

Conclusions

To the best of our knowledge, this paper represents one of the temperature 250 °C, method 1 (initial temperature 100 °C,

first report on the combinatorial synthesis of complex artificial initial time 2 min, rate 20 °C/min, final temperature 290 °C);

(but "natural-based") polyphenols using a fragment-based method 2 (initial temperature 70 °C, initial time 2 min, rate 20

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 9

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 10 of 20

ARTICLE

Organic and Biomolecular Chemistry

°C/min, final temperature 260 °C). The data are reported as (E)-3-(4-(allyloxy)-3-methoxyphenyl)acrylic acid 13 . A solution

View Article Online

follow: retention time (Rt, min), m/z values and the abundance of trans-ferulic acid (6.00 g, 30.9 mmol) ^Din^Od^I:r¹y^0.1M⁰³e⁹C^/CN^7O(1^B0⁰0²¹m⁸²L^H)

relative. Only m/z > 5 are reported. HRMS: samples were was treated with K₂CO₃(10.3 g, 74.16 mmol) and allylbromide

analysed with a Synapt G2 QToF mass spectrometer. MS signals (8.8 mL, 102 mmol). After stirring for 18 h at 70 °C, the resulting

were acquired from 50 to 1200 m/z in either ESI positive or suspension was filtered through a celite cake washing with

negative ionization mode. Column chromatography was done MeCN. After evaporation, the obtained allyl (E)-3-(4-(allyloxy)-

with the "flash" methodology by using 220–400 mesh silica. 3-methoxyphenyl)acrylate was directly dissolved in MeOH (150

Petroleum ether (40–60 °C) is abbreviated as PE. All reactions mL) and treated with 1 N KOH aqueous solution (62 mL, 61.8

employing dry solvents were carried out under nitrogen. mmol). After stirring for 24 h at 60 °C and evaporation to

Extractions were always repeated three times and organic reduced volume, the crude was treated with 1 N NaOH aqueous

extracts were always dried over Na₂SO₄and filtered before solution and extracted with AcOEt. The aqueous phase was

evaporation to dryness.

acidified with 12 N HCl (final pH = 3) and extracted with AcOEt.

4-allyloxybenzoic The organic extracts were washed with saturated brine and

Compounds 3 ^23,

4 ²⁵

5 ³⁹

10 ⁴⁰

12 ^41,

acid,⁴³

4-allyloxy-3-methoxybenzaldehyde,⁴⁴

acid,⁴⁵

and

E-3,4- evaporated. The resulting crude was triturated with Et₂O to give

4- pure (E)-3-(4-(allyloxy)-3-methoxyphenyl)acrylic acid as white

bis(allyloxy)phenylpropenoic

pivaloyloxybenzaldehyde⁴⁶were prepared by the reported solid (6.65 g, 96%). The other spectroscopic and analytical data

methods.

were in agreement with those reported.⁴⁵

Due to a tendency to partially degrade, the peptidomimetics 4-allyloxyaniline. A solution of 4-nitrophenol (5.00 g, 35.9

were in most cases fully characterized and stored in the mmol) in dry MeCN (70 mL) was treated with K₂CO₃(12.4 g, 89.7

acetylated form

2 and then deprotected shortly before use mmol) and allylbromide (4.7 mL, 53.9 mmol). After stirring for

through procedure B, checking the purity by ¹H NMR and HPLC. 18 h at 70 °C, the resulting suspension was filtered through a

4-((tert-Butyldimethylsilyl)oxy)aniline 9. A solution of p- celite cake washing with MeOH. After evaporation, the crude

aminophenol (1.00 g, 9.16 mmol) in dry CH₂Cl₂(30 mL) was was treated with saturated aqueous NH₄Cl and extracted with

treated with imidazole (1.25 g, 18.3 mmol) and tert- Et₂O in order to completely remove the salts. The organic

butyldimethylsilyl chloride (2.07 g, 13.7 mmol). After stirring for extracts were washed with saturated brine and evaporated. The

3 h at r.t. a purple suspension was obtained. It was treated with obtained 1-(allyloxy)-4-nitrobenzene was directly dissolved in

saturated aqueous NaHCO₃and extracted with CH₂Cl₂. After EtOH (70 mL) and treated with Fe powder (16.0 g, 287.2 mmol)

evaporation and chromatography (PE / AcOEt 8:2), pure

9

and a solution of NH₄Cl (7.68 g, 143.6 mmol) in deionized H₂O

(1.880 g, 92%) was obtained as a colorless liquid. δ_H(300 MHz, (28 mL). After stirring for 18 h at 75 °C, the resulting black

CDCl₃, 25 °C): 6.68-6.54 (AA'XX' system, 4 H); 3.41 (br s, 2 H), suspension was filtered through a celite cake washing with

0.97 (s, 9 H), 0.15 (s, 6 H). The other spectroscopic and analytical MeOH. After evaporation, the crude was treated with saturated

data were in agreement with those reported.⁴⁷

4-((tert-Butyldimethylsilyl)oxy)phenyl isocyanide 8. A solution extracts were washed with saturated brine and evaporated. The

of 4-((tert-butyldimethylsilyl)oxy)aniline (1.00 g, 4.48 mmol) resulting crude 4-allyloxyaniline (light brown oil) was used in the

aqueous NaHCO₃and extracted with AcOEt. The organic

9

in dry CH₂Cl₂(45 mL) was treated with formic acid (203 µL, 5.38 next step without further purification. δ_H(300 MHz, d6-DMSO,

mmol), 4-dimethylaminopyridine (DMAP) (101 mg, 0.90 mmol) 25 °C): 12.23 (s, 1 H, OH); 7.52 (d, J 15.9, 1 H, ArCH=CH); 7.33 (d,

and, finally, dicyclohexylcarbodiimide (DCC) (1.017 g, 4.93 J 2.1, 1 H, 1 H, H ortho to OMe); 7.18 (dd, J 8.4, 1.8, 1 H, H para

mmol). After 3.5 h at r.t., the resulting suspension was treated to OMe); 6.98 (d, J 8.4, 1 H, H meta to OMe); 6.45 (d, J 15.9, 1

with additional formic acid (51 µL, 1.34 mmol) and DCC (185 mg, H, ArCH=CH); 6.04 (ddt, J 17.2, 10.6, 5.4, 1 H, CH=CH₂); 5.40 (dq,

0.90 mmol). After further 1.5 h, the suspension was filtered J 17.3, 1.6, 1 H, CH=CHH); 5.27 (dq, J 10.6, 1.5, 1 H, CH=CHH);

through a celite cake washing with CH₂Cl₂. After evaporation 4.60 (dt, J 5.4, 1.5, 2 H, CH₂O); 3.82 (s, 3 H, OCH₃). The

and chromatography (CH₂Cl₂/ acetone 95:5), N-(4-((tert- spectroscopic and analytical data were in agreement with those

butyldimethylsilyl)oxy)phenyl)formamide⁴⁸was

obtained reported.⁴⁷

(83%). 172 mg (0.65 mmol) of this formamide was dissolved in 4-allyloxyphenethylamine. A solution of tyramine (1.50 g,

dry CH₂Cl₂(2.5 mL), cooled to −15 °C, and treated with Et₃N (272 11.00 mmol) in dioxane / H₂O (22 mL, 3:1) was treated at r.t.

µL, 1.95 mmol) and trichloromethyl chloroformate with triethylamine (1.53 mL, 11.00 mmol) and di-tert-butyl

(diphosgene) (46 µL, 0.39 mmol). The temperature was allowed dicarbonate (2.40 g, 11.00 mmol). After 2 h the solution was

to reach 0°C during 1 h and the mixture further stirred for 30 concentrated under vacuum, and the residue was poured into a

min. Then, the reaction was quenched with saturated aqueous mixture of 5% aq (NH₄)H₂PO₄and 1 M HCl and extracted with

NaHCO₃and extracted with CH₂Cl₂. The organic extracts were CH₂Cl₂. The organic extracts were washed with saturated brine

washed

with

saturated

brine,

evaporated

and and evaporated. The resulting N-Boc-tyramine was diluted in

chromatographed (PE / CH₂Cl₂70:30) to give pure

8

as a dry DMF (26 mL) and treated with Cs₂CO₃(4.70 g, 14.4 mmol)

pearlaceous oil (152 mg, 95% from formamide). δ_H(300 MHz, and allylbromide (1.3 mL, 14.1 mmol). After stirring for 4 h at 50

CDCl₃, 25 °C): 7.25 (d, J 8.7, 2 H, ); 6.81 (d, 2 H, J 8.7); 0.98 (s, 9 °C, the mixture was poured in saturated aqueous NH₄Cl and

H), 0.21 (s, 3 H). The other spectroscopic and analytical data extracted with Et₂O. The organic extracts were washed with

were in agreement with those reported.⁴⁸

saturated brine and evaporated. Then, the crude was dissolved

in dry CH₂Cl₂(10 mL) at 0 °C and treated with trifluoroacetic acid

10 | J. Name., 2012, 00, 1-3

This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

Page 11 of 20

Organic & Biomolecular Chemistry

Please do not adjust margins

Organic and Biomolecular Chemistry

(5 mL). After stirring for 3 h at r.t., the solution was evaporated N-(4-allyloxyphenyl)formamide.

to dryness, taken up with 1 M aqueous NaOH and extracted allyloxyaniline⁵¹(499 mg, 3.35 mmol) in dry CH₂Cl₂(17 mL) at 0

with CH₂Cl₂to give pure 4-allyloxyphenethylamine as yellow oil °C was treated with formic acid (152 µL, 4.02 mmol), 4-

(1.72 g, 88% from tyramine). R_f= 0.33 (CH₂Cl₂/MeOH 15:1 + 1% dimethylaminopyridine (DMAP) (82 mg, 0.67 mmol) and, finally,

of Et₃N). δ_H(300 MHz, CDCl₃, 25 °C): 7.10 (d, J 8.4, 2 H, H meta dicyclohexylcarbodiimide (DCC) (760 mg, 3.69 mmol). After 2 h

to OAll); 6.86 (d, J 8.4, 2 H H ortho to OAll); 6.05 (ddt, J 17.0, at r.t., the resulting suspension was filtered through a celite

10.6, 5.3, 1 H, CH=CH₂); 5.40 (dq, J 17.0, 1.5, 1 H, CH=CHH); 5.27 cake washing with Et₂O + 2% of CH₂Cl₂. After evaporation and

ARTICLE

A

solution

of

4-

View Article Online

DOI: 10.1039/C7OB02182H

(dq, J 10.6, 1.2, 1 H, CH=CHH); 4.51 (dt, J 5.4, 1.2, 2 H, CH₂O); chromatography

(CH₂Cl₂

/

AcOEt

7:1),

N-(4-

2.92 (t, J 6.8, 2 H, CH₂N); 2.68 (t, J 6.8, 2 H, ArCH₂). δ_C(75 MHz, allyloxyphenyl)formamide was obtained (564 mg, 95%) as

CDCl₃, 25 °C): 156.8, 133.1, 131.4, 129.4 (x2), 117.1, 114.4 (x2), yellow solid. M.p.: 50.9−52.1 °C (CH₂Cl₂). R_f= 0.45 (PE / AcOEt

68.5, 49.3, 43.1, 38.4. IR: 

_max/cm^-13373, 3029, 2926, 2857, 1:1). δ_H(300 MHz, CDCl₃, 25 °C)(two conformers in about 1:1

1715, 1648, 1610, 1582, 1509, 1457, 1424, 1382, 1362, 1297, ratio are visible): 8.50 (0.5 H, d, J 11.6, CHO of 1 conformer);

1237, 1221, 1177, 1154, 1111, 1069, 1021, 996, 924, 818, 752, 8.34 (0.5 H, d, J 1.8, 0.5 H, CHO of 1 conformer); 7.59 (0.5 H,

644, 617. GC-MS (method 1) t_R7.17 min, m/z (%) 148 ([M- broad s, NH of 1 conformer); 7.44 (1 H, d, J 9.0, ArCH of 1

CH₂NH₂]⁺, 7.4), 107 (17), 91 (7.2), 79 (6.4), 78 (7.4), 77 (13), 55 conformer); 7.12 (0.5 H, broad s, NH of 1 conformer); 7.03 (1 H,

(7.6), 52 (7.5), 51 (9.7), 42 (5.0), 41 (100), 39 (35). The other d, J 9.0, ArCH of 1 conformer); 6.91 (1 H, d, J 9.0, ArCH of 1

spectroscopic and analytical data were in agreement with those conformer); 6.89 (1 H, d, J 9.0, ArCH of 1 conformer); 6.05 (1 H,

reported.⁴⁹

ddt, J 10.5 17.2 (d), 5.3 (t), CH=CH₂); 5.46-5.36 (1 H, m,

3-allyloxybenzylamine. A solution of 3-allyloxybenzyl alcohol⁵⁰CH=CHH); 5.33-5.26 (1 H, m, CH=CHH); 4.55-4.50 (2 H, m,

(2.66 g, 16.1 mmol) in dry CH₂Cl₂(55 mL) was cooled at −15 °C CH₂CH=CH₂). δ_C(75 MHz, CDCl₃, 25 °C): 163.3, 159.3 (C=O),

and treated with Et₃N (2.9 mL, 20.9 mmol) and mesyl chloride 156.4, 155.5, 130.2, 129.8 (arom. quat.), 133.0, 132.9 (CH=CH₂),

(1.5 mL, 19.3 mmol). After stirring for 4 h at −15 °C, the solvent 121.7, 121.2, 115.6, 114.9 (ArCH), 117.8, 117.6 (CH=CH₂), 69.0,

was evaporated and the obtained mesylate was directly 68.9 (CH₂O). IR: 

_max/cm^-13297, 3269, 3208, 3144, 3106, 3084,

dissolved in dry DMF (23 mL) and treated NaN₃(2.30 g, 33.8 3020, 2977, 2941, 2925, 2869, 2803, 2771, 1657, 1644, 1612,

mmol). After stirring for 3 days at r.t., the mixture was poured 1547, 1507, 1465, 1426, 1409, 1391, 1370, 1340, 1327, 1303,

in H₂O and extracted with Et₂O. The organic extracts were 1254, 1229, 1177, 1151, 1123, 1111, 1062, 1013, 1002, 940,

washed with saturated brine (× 5) and evaporated. The crude 931, 873, 839, 822, 749, 737, 709, 648, 633. GC-MS (method 2)

was purified by chromatography (PE / CH₂Cl₂from 8:2 to 7:3) to t_R7.88 min, m/z (%) 177 (43) [M]⁺, 137 (8.5), 136 (100), 109 (11),

give pure 3-allyloxybenzyl azide as pale yellow oil (2.63 g, 86% 108 (99), 81 (6.4), 80 (50), 65 (8.6), 63 (5.2), 54 (5.5), 53 (26), 52

from 3-allyloxybenzyl alcohol). R_f= 0.29 (PE / CH₂Cl₂8:2). δ_H(300 (16), 41 (37), 39 (27). m/z (ESI+) 178.0867 (M + H⁺). C₁₀H₁₂O₂N

MHz, CDCl₃, 25 °C): 7.29 (mc) (1 H, m, ArCH); 6.93-6.86 (3 H, m, requires 178.0868.

ArCH); 6.06 (1 H, ddt, J 10.5, 17.2, 5.3 (t), CH=CH₂); 5.42 (1 H, 4-allyloxyphenyl isocyanide.

A

solution of N-(4-

dq, J 17.2(d), 1.5 (q), CH=CHH); 5.30 (1 H, dq, J 10.5 (d), 1.5 (q), allyloxyphenyl)formamide (130 mg, 0.734 mmol) in dry CH₂Cl₂

CH=CHH); 4.55 (2 H, dt, J 5.3 (d), 1.5 (t), CH₂CH=CH₂); 4.30 (2 H, (7 mL) was treated with Et₃N (470 µL, 3.37 mmol) and cooled at

s, CH₂N₃). δ_C(75 MHz, CDCl₃, 25 °C): 158.9, 136.9 (arom. quat.), −30 °C. Then POCl₃(103 µL, 1.10 mmol) was added dropwise.

133.1 (CH=CH₂), 129.9, 120.6, 114.6, 114.5 (ArCH), 117.8 After stirring for 1 h at −30 °C, the cold mixture was poured in

(CH=CH₂), 68.8 (CH₂O), 54.7 (CH₂N). IR: 

_max/cm^-13064, 2925, saturated aqueous NaHCO₃and extracted with Et₂O. The

2870, 2094, 1649, 1599, 1586, 1489, 1448, 1424, 1342, 1263, organic extracts were washed with saturated brine and

1157, 1098, 1027, 994, 927, 878, 854, 783, 762, 695, 650. GC- evaporated. The crude was purified by chromatography (PE /

MS (method 1) t_R5.02 min, m/z (%) 189 (1.2) [M]⁺, 120 (5.5), 92 Et₂O 15:1) to give pure 4-allyloxyphenyl isocyanide as green oil

(6.2), 91 (6.7), 79 (5.5), 78 (9.2), 77 (8.1), 65 (20), 64 (6.8), 63 (107 mg, 91%). R_f= 0.30 (PE / Et₂O 15:1). δ_H(300 MHz, CDCl₃, 25

(10), 51 (9.5), 50 (6.2), 41 (100), 39 (46), 38 (7.0). A solution of °C): 7.30 (2 H, d, J 8.9, ArCH); 6.88 (2 H, d, J 8.9, ArCH); 6.03 (1

this azide (2.63 g, 13.9 mmol) in dry DMF (40 mL) cooled to 0 °C H, ddt, J 10.5, 17.3 (d), 5.3 (t), CH=CH₂); 5.41 (1 H, dq, J 17.3 (d),

was treated with PMe₃(1 M in toluene, 15.3 mL, 15.3 mmol). 1.5 (q), CH=CHH); 5.32 (1 H, dq, J 10.5 (d), 1.5 (q), CH=CHH); 4.55

When the gas evolution ceased, the mixture was warmed up to (2 H, dt, J 5.3 (d), 1.5 (t), CH₂CH=CH₂). δ_C(75 MHz, CDCl₃, 25 °C):

r.t. and stirred for 2 h. Then H₂O (1 mL, 55.6 mmol) was added 162.5 (NC), 158.8, 119.6 (broad) (arom. quat.), 132.3 (CH=CH₂),

and the reaction was further stirred for 2 h at r.t. After 127.7, 115.3 (ArCH), 118.3 (CH=CH₂), 69.0 (CH₂O). IR: 

_max/cm^-1

evaporation, the mixture was treated with saturated aqueous 3675, 3082, 2986, 2901, 2123, 1735, 1648, 1605, 1584, 1502,

Na₂CO₃and extracted with AcOEt. The organic extracts were 1456, 1423, 1409, 1383, 1298, 1247, 1230, 1192, 1164, 1109,

washed with saturated brine and evaporated to give crude 3- 1067, 1048, 1015, 995, 928, 830, 739, 700, 647, 618. GC-MS

allyloxybenzylamine, that was not purified, but used as such for (method 2) t_R5.67 min, m/z (%) 159 (92) [M]⁺, 158 (19), 144 (19),

the Ugi reaction. It was just controlled at ¹H NMR, that showed 132 (7.0), 131 (8.0), 130 (19), 119 (29), 103 (5.9), 102 (11), 91

a purity > 95%. δ_H(300 MHz, CDCl₃, 25 °C): 7.24 (1 H, t, J 8.1); (11), 90 (12), 76 (7.7), 75 (8.6), 64 (19), 63 (13), 41 (100), 39 (19).

6.92-6.87 (2 H, m); 6.85-6.77 (1 H, m); 6.06 (1 H, ddt, J 10.5, 17.3 m/z (ESI+) 160.0769 (M + H⁺). C₁₀H₁₀ON requires 160.0762.

(d), 5.3 (t), CH=CH₂); 5.45 (1 H, dq, J 17.3 (d), 1.5 (q), CH=CHH); N-(4-(2-(2-(allyloxy)ethoxy)ethoxy)phenyl)formamide. Known

5.28 (1 H, dq, J 10.5 (d), 1.5 (q), CH=CHH); 4.55 (2 H, dt, J 5.3 (d), 1-(2-(2-(allyloxy)ethoxy)ethoxy)-4-nitrobenzene was prepared

1.5 (t), CH₂CH=CH₂); 3.84 (2 H, s, CH₂NH₂).

according to literature procedures.^{52, 53}This compound (760

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 11

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 12 of 20

ARTICLE

Organic and Biomolecular Chemistry

mg, 2.84 mmol) was dissolved in EtOH (33 mL) and treated with 1127, 1108, 1058, 995, 923, 883, 832, 724, 681,_Vi6_ew41_Ar._ticG_leC_O-_nM_linS_e

Fe powder (1.27 g, 22.7 mmol) and a solution of NH₄Cl (607 mg, (method 1) t_R7.50 min, m/z (%) 159 (1.1)^D, 1^O0^I:2¹⁰(^.6¹⁰.9³)⁹,^/8^C5^7O(^B7⁰.1²)¹,⁸7^2H3

11.4 mmol) in deionized H₂O (6 mL). After stirring for 2 h at 75 (6.0), 71 (5.2), 45 (11), 43 (18), 41 (100), 39 (13). m/z (ESI+):

°C, the resulting black suspension was filtered through a celite 248.1288 (M + H⁺). C₁₄H₁₈O₃N requires 248.1287.

cake washing with MeOH. After evaporation, the crude was (R,S)-N-(4-Acetoxyphenyl)-N-(1-(4-acetoxyphenyl)-2-(tert-

treated with saturated aqueous NaHCO₃and extracted with butylamino)-2-oxoethyl)propionamide 2a

AcOEt. The organic extracts were washed with saturated brine aldehyde 4²⁵(228 mg, 2.0 mmol) in dry methanol (6.7 mL) was

and evaporated. The resulting crude

3-(2-(2- treated with amine 3^{23, 24}(302 mg, 2.0 mmol), propionic acid

. A solution of

(allyloxy)ethoxy)ethoxy)aniline was directly treated with ethyl (150 µL, 2.0 mmol), and tert-butyl isocyanide (225 µL, 2.0

formate (4 mL) and stirred at 60 °C for 6 days. After evaporation, mmol). The solution was stirred at r.t. for 5 h. Then the solvent

the crude was purified by chromatography (PE / AcOEt 1:1) to was evaporated and the crude purified by chromatography (PE

give pure N-(4-(2-(2-(allyloxy)ethoxy)ethoxy)phenyl)formamide / AcOEt 1:1) to give pure 2a as a slightly brown solid (665 mg,

as

brown

oil

(506

mg,

67%

from

1-(2-(2- 73%). M.p. = 144.6-146.8 °C. R_f= 0.29 (PE / AcOEt 40:60). δ_H(300

(allyloxy)ethoxy)ethoxy)-4-nitrobenzene). R_f= 0.24 (PE / AcOEt MHz, CDCl₃, 25 °C): 7.13 (2 H, d, J 8.5, ArCH from aldehyde); 6.92

1:1). δ_H(300 MHz, CDCl₃, 25 °C)(two conformers in about 1:1 (2 H, d, J 8.5, ArCH from aldehyde); 7.05-6.85 (2 H, broad m,

ratio are visible): 8.50 (0.5 H, d, J 11.6, CHO of 1 conformer); ArCH from amine) (NOTE: the other 2 ArCH from amine give a

8.33 (0.5 H, d, J 1.8, CHO of 1 conformer); 7.46 (0.5 H, broad s, very broad signal from 7.50 to 7.00), 5.99 (1H, 1 H, CH), 5.72

NH of 1 conformer); 7.43 (1 H, d, J 9.0, ArCH of 1 conformer); (1H, s, NH), 2.25 (6H, s, CH₃CO), 2.12-2.00 (2H, m, CH₂CH₃), 1.34

7.12 (0.5 H, broad s, NH of 1 conformer); 7.01 (1 H, d, J 9.0, ArCH (9H, s, (CH₃)₃)C), 1.04 (3 H, t, J= 7.4, CH₂CH₃). δ_C(75 MHz, CDCl₃,

of 1 conformer); 6.91 (1 H, d, J 9.0, ArCH of 1 conformer); 6.89 25 °C): 174.3 (C=O), 169.0 (C=O), 168.8 (C=O), 168.7 (C=O),

(1 H, d, J 9.0, ArCH of 1 conformer); 5.92 (1 H, ddt, J 10.4, 17.2 150.5, 150.0, 137.2, 132.3 (quat.), 131.5, 131.3, 121.8, 121.4

(d), 5.7 (t), CH=CH₂); 5.28 (1 H, dq, J 17.2 (d), 1.5 (q), CH=CHH); (ArCH), 64.3 (CH), 51.5 (C(CH₃)₃), 28.5 (C(CH₃)₃), 28.3 (CH₂CH₃),

5.19 (1 H, dq, J 10.5 (d), 1.5 (q), CH=CHH); 4.15-4.09 (2 H, m, 21.0 (CH₃CO), 9.3 (CH₂CH₃). IR: 

_max/cm^-13339, 3234, 3078,

CH₂O); 4.04 (2 H, dt, J 5.7(d), 1.5 (t), OCH₂CH=CH₂); 3.89-3.83 (2 2976, 1759, 1681,1636, 1551, 1504, 1459, 1418, 1390, 1366,

H, m, CH₂O); 3.76-3.69 (2 H, m, CH₂O); 3.67-3.60 (m, 2 H, CH₂O). 1305, 1270, 1251, 1209, 1186, 1166, 1160, 1105, 1099, 1044,

δ_C(75 MHz, CDCl₃, 25 °C):

 = 163.1, 159.3 (C=O), 156.4, 155.4, 1012, 958, 941, 910, 859, 848, 813, 784, 775, 744, 735, 722, 656,

130.4, 129.9 (arom. quat.), 134.3 (CH=CH₂), 121.5, 121.1, 115.4, 632. m/z (ESI+): 455.2180 (M + H⁺). C₂₅H₃₁O₆N₂requires

114.6 (ArCH), 117.2 (CH=CH₂), 72.0, 70.6, 69.5, 69.2, 67.5, 67.4 455.2182.

(CH₂O). IR: 

_max/cm^-13676, 3274, 3130, 3071, 2871, 1669, 1602, (R,S)-N-(4-Hydroxyphenyl)-N-(2-(tert-butylamino)-1-(4-

1536, 1509, 1455, 1412, 1351, 1290, 1234, 1176, 1127, 1090, hydroxyphenyl)-2-oxoethyl)propionamide 1a. A solution of

1062, 994, 923, 872, 827, 726, 643, 633. GC-MS (method 1) t_Rcompound 2a (137 mg, 0.30 mmol) in tetrahydrofuran (2.25 mL)

9.19 min, m/z (%) 163 ([M−OCH₂CH₂OAllyl]⁺, 1.0) 108 (8.7), 87 was treated, at r.t., with 1 M acqueous LiOH (0.78 µL, 0.78

(8.8), 85 (9.2), 80 (8.1), 65 (9.7), 53 (7.0), 45 (9.5), 44 (5.4), 43 mmol). The solution became yellow. After 20 h, the reaction not

(21), 41 (100), 39 (10). m/z (ESI+): 266.1392 (M + H⁺). C₁₄H₂₀O₄N being yet complete, other 0.39 µL of LiOH solution were added.

requires 266.1392.

N-(4-(2-(2-(allyloxy)ethoxy)ethoxy)phenyl) isocyanide.

After other 20 h, the reaction was worked out with a 1 M

NaH₂PO₄solution and extracted with AcOEt. After evaporation

A

solution

of

N-(4-(2-(2- of the organic phase, two consecutive chromatographies (first

(allyloxy)ethoxy)ethoxy)phenyl)formamide (374 mg, 1.41 PE / AcOEt 3:7; then CH₂Cl₂/ MeOH 93:7) afforded pure 1a as a

mmol) in dry CH₂Cl₂(7 mL) was treated with Et₃N (590 µL, 4.23 white solid (85 mg, 76%). M.p. = 203.4-204.1 °C. R_f= 0.24 (PE /

mmol) and cooled at 0 °C. Then diphosgene (103 µL, 0.85 mmol) AcOEt 30:70). δ_H(300 MHz, DMSO-d6, 50 °C): 9.22 (1 H, br s,

was added dropwise. After stirring for 1 h at 0 °C, the cold OH), 9.16 (1 H, br s, OH); 7.28 (1 H, s, NH); 6.81 (2 H, d, J 8.5,

mixture was poured in saturated aqueous NaHCO₃and ArCH from aldehyde); 6.49 (2 H, d, J 8.5, ArCH from aldehyde);

extracted with Et₂O. The organic extracts were washed with 6.70-6.45 (2 H, broad m, ArCH from amine) (NOTE: the other 2

saturated brine and evaporated. The crude was purified by ArCH from amine give a very broad signal from 7.30 to 6.80),

chromatography (PE / AcOEt 8:2) to give pure N-(4-(2-(2- 5.87 (1H, s, CH), 2.01-1.81 (2H, m, CH₂CH₃), 1.23 (9H, s,

(allyloxy)ethoxy)ethoxy)phenyl) isocyanide as yellow oil (307 (CH₃)₃)C), 0.89 (3 H, t, J= 7.4, CH₂CH₃). δ_C(75 MHz, DMSO-d6, 50

mg, 88%). R_f= 0.34 (PE / AcOEt 8:2). δ_H(300 MHz, CDCl₃, 25 °C): °C): 172.6 (C=O), 169.5 (C=O), 156.1, 155.8, 131.5, 131.1 (quat.),

7.30 (2 H, d, J 9.0, ArCH); 6.89 (2 H, d, J 9.0, ArCH); 5.92 (1 H, 130.9, 126.1, 114.5, 114.2 (ArCH), 63.0 (CH), 49.9 (C(CH₃)₃), 28.3

ddt, J 10.4, 17.2 (d), 5.7 (q), CH=CH₂); 5.28 (1 H, dq, J 17.2 (d), (C(CH₃)₃), 27.3 (CH₂CH₃), 9.2 (CH₂CH₃). IR: 

_max/cm^-13274, 3234,

1.6 (q), CH=CHH); 5.19 (1 H, dq, J 10.5 (d), 1.2 (q), CH=CHH); 2969, 1661, 1614, 1593, 1511, 1452, 1393, 1365, 1258, 1221,

4.17-4.11 (2 H, m, CH₂O); 4.03 (2 H, dt, J 5.7 (d), 1.3 (t), 1174, 1096, 1044, 1023, 960, 845, 816, 780, 740, 633. m/z

OCH₂CH=CH₂); 3.89-3.85 (2 H, m, CH₂O); 3.75-3.69 (2 H, m, (ESI+): 371.1976 (M + H⁺). C₂₁H₂₇O₄N₂requires 371.1971. HPLC

CH₂O); 3.66-3.60 (2 H, m, CH₂O). δ_C(75 MHz, CDCl₃, 25 °C): 162.5 (see supplementary information) showed a purity of 99.5%.

(NC), 159.1, 119.3 (broad) (arom. quat.), 134.6 (CH=CH₂), 127.6, (R,S)-4-((tert-Butyldimethylsilyl)oxy)-N-(4-((tert-

155.2 (ArCH), 117.1 (CH=CH₂), 72.2, 70.8, 69.5, 69.3, 67.7 butyldimethylsilyl)oxy)phenyl)-N-(2-((4-((tert-

(CH₂O). IR:

1585, 1504, 1453, 1423, 1394, 1352, 1298, 1252, 1194, 1164, phenylethyl)benzamide 11. A solution of amine



_max/cm^-13676, 3078, 2871, 2122, 1741, 1646, 1605, butyldimethylsilyl)oxy)phenyl)amino)-2-oxo-1-

9

(224 mg, 1.00

12 | J. Name., 2012, 00, 1-3

This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

Page 13 of 20

Organic & Biomolecular Chemistry

Please do not adjust margins

Organic and Biomolecular Chemistry

ARTICLE

mmol) in dry CH₂Cl₂(10.0 mL) was treated at r.t. with treated with PdCl₂(PPh₃)₂(0.025 equiv for allyl group) and

View Article Online

benzaldehyde (102 µL, 1.00 mmol) and anhydrous MgSO₄(100 ammonium formate (2.2 equiv for allyl gr^Do^Ou^Ip^:)¹⁰a^.1t⁰8³0^9/°^CC^7Ofo^Br⁰2²¹h⁸²i^Hn

mg) and stirred overnight. After filtration of MgSO₄and a sealed flask. Then, the crude was diluted with AcOEt, washed

evaporation, the residue was taken up in MeOH (5.0 mL), added with saturated aqueous NaHCO₃. After evaporation, the residue

with freshly activated powdered 3 Å molecular sieves (50 mg), was eluted from a column of silica gel with the suitable eluent.

and finally treated with acid 10 (252 mg, 1.00 mmol) and (R,S)-(E)-N-Benzyl-N-(2-(tert-butylamino)-1-(4-

isocyanide

8

(234 mg, 1.00 mmol). After 17 h, the mixture was hydroxyphenyl)-2-oxoethyl)-3-(4-hydroxy-3-

Following the general

filtered, evaporated to dryness and chromatographed (PE / methoxyphenyl)acrylamide 1c

.

AcOEt 60:40) to give pure 11 as a yellow-brown solid (200 mg, procedure A, a mixture of aldehyde 12 (113 mg, 0.70 mmol),

25%). M.p: = 156.7-157.3 °C. R_f= 0.58 (PE / AcOEt 6:4). δ_H(300 benzylamine (80 µL, 0.77 mmol), acid 13 (180 mg, 0.77 mmol),

MHz, CDCl₃, 25 °C): δ 8.19 (1 H, s, NH), 7.35 (2 H, d, J 9.0, ArH), t-butyl isocyanide (87 µL, 1.19 mmol) and 3 Å molecular sieves

7.32-7.18 (7 H, m), 6.85 (2 H, broad d, J= 7.7 Hz, ArH), 6.74 (2 H, (32 mg) was stirred for 3 days at r.t. After work-up and

d, J= 8.8 Hz, ArH), 6.56 (2 H, d, J= 8.7 Hz, ArH), 6.50 (2 H, d, J= purification (PE / AcOEt 50:50) compound 14c was obtained

8.9 Hz, ArH), 6.34 (1 H, s, CH), 0.96, 0.92, 0.91 (3 x 9 H, 3 s, pure as white foam (275 mg, 69%). Then a mixture of 14c (255

(CH₃)₃C); 0.16, 0.11, 0.08 (3 x 6 H, 3 s, (CH₃Si). δ_C(75 MHz, CDCl₃, mg, 0.45 mmol), PdCl₂(PPh₃)₂(16 mg, 0.023 mmol) and

25 °C, TMS): 171.1, 168.1 (C=O), 156.9, 154.6, 152.3, 135.0, ammonium formate (124 mg, 1.98 mmol) was stirred for 2 h at

134.5, 131.50, 131.2 (quat.), 131.4 (x2), 130.8 (x2), 130.2 (x2), 80 °C. After work-up and purification (chromatography with PE

128.6, 128.5 (x2), 121.7 (x2), 120.2 (x2), 120.0 (x2), 119.1 (x2) / AcOEt 3:4, followed by treatment with active coal) compound

(ArCH), 67.4 (CH), 25.69, 25.64, 25.56 (C(CH₃)₃), 18.38 (C(CH₃)₃), 1c was obtained pure as white solid (177 mg, 80%). M.p. = 125

18.24, 18.19, 18.14 (quat. C t-Bu), −4.48 (x2), −4.54 (CH₃Si). IR: °C with decomposition. R_f= 0.19 (PE / AcOEt 50:50). δ_H(300



_max/cm^-13260, 3201, 3075, 2957, 2930, 2896, 2858, 1689, MHz, DMSO-d6, 90 °C): δ 9.06 (2 H, s, OH), 7.43 (1 H, broad s,

1617, 1604, 1551, 1505, 1472, 1462, 1410, 1389, 1362, 1341, NH), 7.41 (1 H, d, J= 15.0, ArCH=CH), 7.20-6.87 (9 H, m, ArH,

1253, 1201, 1166, 1103, 1080, 1052, 1006, 966, 908, 831, 803, ArCH=CH), 6.80-6.60 (1 H, broad signal, ArCH=CH), 6.76 (1 H, d,

777, 764, 735, 716, 698, 666, 638, 623. m/z (ESI+): 797.4187 (M J 8.1, H meta to OMe), 6.67 (2 H, d, J 8.7, H ortho to OH), 6.00

+ H⁺). C₄₅H₆₅O₅N₂Si₃requires 797.4201.

(R,S)-4-Hydroxy-N-(4-hydroxyphenyl)-N-(2-((4-

(1 H, broad s, CHN), 4.87 (1 H, d, J 16.7, CHHPh), 4.55 (1 H, d, J

16.7, CHHPh), 3.78 (3 H, s, OCH₃), 1.26 (9 H, s, C(CH₃)₃). δ_C(75

hydroxyphenyl)amino)-2-oxo-1-phenylethyl)benzamide 1b. A MHz, DMSO-d6, 25 °C) (note: at this temperature, 2 conformers

solution of compound 2a (117 mg, 0.15 mmol) in are visible and thus most signals are doubled): 169.6, 169.2,

tetrahydrofuran (1.2 mL) was treated, at r.t., with 1 M acqueous 167.1, 166.9 (C=O), 156.9, 156.8, 148.4, 147.7, 147.6, 140.1,

LiOH (0.59 µL, 0.59 mmol). The solution became orange. After 139.4, 130.0 (quat.), 141.7 (ArCH=CH), 130.4, 127.8, 127.4,

22 h the solution was evaporated, taken up with MeOH, and 126.8, 126.4, 126.2, 126.1, 122.3, 121.9, 116.3, 114.9, 110.6

treated with previously washed Amberlyst 15 acid resin until pH (ArCH), 115.4 (ArCH=CH), 62.8, 60.2 (CHN), 55.5, 55.4 (OCH₃),

= 7. The resin was filtered off and the solution evaporated to 50.2 (C(CH₃)₃), 48.0 (CH₂Ph), 28.3, 28.1 (C(CH₃)₃). IR: 

_max/cm^-1

dryness and chromatographed (PE / AcOEt 4:6 + 2% EtOH) to 3283, 2967, 1641, 1589, 1511, 1452, 1429, 1364, 1265, 1203,

give pure 1b as a white solid (54 mg, 81%). M.p. = 177.7-178.2 1171, 1123, 1080, 1030, 976, 947, 891, 865, 837, 814, 726, 696.

°C °C. R_f= 0.34 (PE / AcOEt 40:60 + 2% EtOH). δ_H(300 MHz, m/z (ESI+): 489.2396 (M + H⁺). C₂₉H₃₃O₅N₂requires 489.2389.

DMSO-d6, 25 °C): δ 9.98, 9.68, 9.24 (3 x 1 H, 3 s, OH), 7.40 (2 H, HPLC (see supplementary information) showed a purity of 99%.

d, J= 9.0, ArH), 7.24–7.10 (5 H, m, ArH+NH), 7.07 (2 H, d, J= 9.3, (R,S)-(E)-N-Benzyl-N-(2-(tert-butylamino)-1-phenyl-2-

ArH), 6.69 (2 H, d, J= 9.0, ArH), 6.51 (2 H, d, J= 8.7, ArH), 6.33 (2 oxoethyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide

1e.

H, broad d, J= 8.4, ArH), 6.26 (1H, s, CH). δ_C(75 MHz, DMSO-d6, Following the general procedure A, a mixture of benzaldehyde

25 °C): 169.8, 168.2 (C=O), 158.1, 155.4, 153.1, 135.2, 132.0, (67 mg, 0.63 mmol), benzylamine (76 µL, 0.69 mmol), (E)-3-(3,4-

131.9, 130.8 (quat.), 130.2 (x4), 127.8 (x 2), 127.7, 127.0 (x2), bis(allyloxy)phenyl)acrylic acid (179 mg, 0.69 mmol), t-butyl

120.6 (x2), 115.0 (x2), 114.2 (x2), 114.0 (x2) (ArCH) 64.9 (CH). IR: isocyanide (78 µL, 0.69 mmol) and 3 Å molecular sieves (28 mg)



_max/cm^-13275, 3234, 1665, 1607, 1509, 1440, 1365, 1223, was stirred for 3 days at r.t. After work-up and purification (PE

1167, 1103, 1081, 831, 761, 729, 698, 626. m/z (ESI+): 455.1604 / AcOEt 75:25) compound 14e was obtained pure as white foam

(M

H⁺). C₂₇H₂₃O₅N₂requires 455.1607. HPLC (see (208 mg, 72%). Then a mixture of 14e (200 mg, 0.37 mmol),

supplementary information) showed a purity of 96%. PdCl₂(PPh₃)₂(12 mg, 0.017 mmol) and ammonium formate (95

+

General procedure for the preparation of polyphenols 1c,e,hi mg, 1.50 mmol) was stirred for 2 h at 80 °C. After work-up and

through allylated derivatives 14c,e,h,i (Method A). A solution purification (two consecutive chromatograhies with PE / AcOEt

of the appropriate aldehyde (1 equiv) in dry EtOH and from 60:40 to 50:50 and a filtration with celite on a sintered

trifluoroethanol in 1:1 ratio (0.26 M) was treated with the funnel) compound 1e was obtained pure as pale yellow solid

amine (1.1 equiv) and molecular sieves (3 Å, 50 mg/mmol). After (101 mg, 59%). M.p. = 120.0 – 121.0 °C (MeOH). R_f= 0.40 (PE /

5 h, the acid (1.1 equiv), and the isocyanide (1.1 equiv) were AcOEt 50:50). δ_H(300 MHz, DMSO-d6, 80 °C) (note: the OH

added. The reaction mixture was stirred at r.t. for 2-4 days, then protons give a very broad signal around 9 ppm): 7.61 (1 H, broad

filtered with celite on a sintered funnel, washed with AcOEt, s, 1 H, NH), 7.38 (1 H, d, J= 15.2, ArCH=CH), 7.30-7.03 (10 H, m,

concentrated and purified by chromatography. A 0.1 M solution ArH), 6.87 (1 H, s, H ortho to OH and CH=CH), 6.79, 6.72 (2 H, AB

of the Ugi product in MeCN under nitrogen atmosphere, was syst., J 8.3, H para to OH and meta to CH=CH), 6.65 (1 H, broad

This journal is © The Royal Society of Chemistry 20xx

J. Name., 2013, 00, 1-3 | 13

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 14 of 20

ARTICLE

Organic and Biomolecular Chemistry

d, J 15.2, ArCH=CH), 6.12 (1 H, broad s, CHN), 4.91, 4.65 (2 H, AB PE/AcOEt) compound 1i was obtained pure as white solid (50

View Article Online

DOI: 10.1039/C7OB02182H

syst., J 17.0, CH₂Ph), 1.24 (9 H, s, C(CH₃)₃). δ_C(75 MHz, DMSO- mg, 68%). M.p. = 150 °C with decomposition. R_f= 0.10 (PE /

d6, 25 °C) (note: at this temperature, 2 conformers are visible AcOEt 50:50). δ_H(300 MHz, DMSO-d6, 25 °C): 9.42 (2 H, s, OH),

and thus most signals are doubled, although one conformer is 9.30 (1 H, s, OH), 7.56 (1 H, s, NH), 7.38 (1 H, d, J 15.3, ArCH=CH),

prevailing, only the peaks of major conformer are reported): 6.88 (1 H, s, H ortho to OMe), 6.83 (2 H, d, J 8.4, H meta to OH),

168.9, 167.1 (C=O), 147.6, 145.3, 139.5, 136.9, 128.2 (quat.), 6.76-6.67 (2 H, m, H meta and para to OMe), 6.70-6.40 (2 H, very

142.5 (ArCH=CH), 128.9 (x2), 128.1 (x2), 127.8 (x2), 127.4, broad signal, H meta to OH), 6.50 (4 H, d, J 8.4, H ortho to OH),

126.2, 125.8 (x2), 120.7, 115.4, 114.1 (ArCH), 115.1 (ArCH=CH), 5.98 (1 H, s, CHN), 5.95 (1 H, d, J 15.3, ArCH=CH), 3.70 (3 H, s,

60.6 (CHN), 54.8 (OCH₃), 50.3 (C(CH₃)₃), 48.1 (CH₂Ph), 28.2 OCH₃), 1.24 (9 H, s, (CH₃)₃C). δ_H(75 MHz, DMSO-d6, 25 °C):

(C(CH₃)₃). IR (ATR):

 = 3276, 3064, 2969, 1640, 1578, 1513, 169.7, 165.4 (C=O), 156.3, 156.0, 148.3, 147.5, 132.0, 130.7,

1451, 1413, 1363, 1278, 1192, 1112, 1080, 1032, 975, 948, 893, 126.2 (quat.), 140.6 (ArCH=CH), 132.0 (x2), 131.2 (x2), 126.1,

845, 810, 753, 730, 696, 617 cm^-1. m/z (ESI-): 457.2143 (M - H⁺). 120.2, 114.5 (x4), 112.2 (ArCH), 116.6 (ArCH=CH), 63.3 (CHN),

C₂₈H₂₉O₄N₂requires 457.2127. HPLC (see supplementary 55.5 (OCH₃), 50.1 (C(CH₃)₃), 28.4 (C(CH₃)₃). IR: 

_max/cm^-13269,

information) showed a purity of 100%.

(R,S)-N-(4-hydroxyphenyl)-N-(1-(4-hydroxyphenyl)-2-((4-

hydroxyphenyl)amino)-2-oxoethyl)-

2966, 2930, 1662, 1640, 1592, 1511, 1450, 1388, 1366, 1258,

1216, 1161, 1121, 1030, 1009, 976, 936, 885, 840, 814, 791, 742,

723, 693, 645, 610. m/z (ESI+): 491.2187 (M + H⁺). C₂₈H₃₁O₆N₂

propanamide

1h.

Following the general procedure A, a mixture of aldehyde 12 requires 491.2182. HPLC (see supplementary information)

(170 mg, 1.05 mmol), 4-allyloxyaniline (173 mg, 1.16 mmol), showed a purity of 98.5%.

propionic acid (87 µL, 1.16 mmol), 4-allyloxyphenyl isocyanide General procedure for the preparation of acetylated

(185 mg, 1.16 mmol) and 3 Å molecular sieves (50 mg) was polyphenols 2d,f,g,,j,l,m,n,o,p,q and 15 (Method B). A solution

stirred for 5 days at r.t. After usual work-up, the crude was of the appropriate aldehyde (1 equiv) in dry EtOH and

diluted with AcOEt, washed with HCl 1 N to remove the excess trifluoroethanol in 1:1 ratio (0.26 M) was treated with the

of amine. Then, the crude was purified (PE / Et₂O 1:2) obtaining amine (1.1 equiv) and molecular sieves (3 Å, 50 mg/mmol). After

compound 14h as white foam (121 mg, 22%). Then a mixture of 5 h, the acid (1.1 equiv), and the isocyanide (1.1 equiv) were

14h (102 mg, 0.20 mmol), PdCl₂(PPh₃)₂(7 mg, 0.01 mmol) and added. The reaction mixture was stirred at r.t. for 2-4 days, then

ammonium formate (83 mg, 1.32 mmol) was stirred for 2 h at filtered with celite on a sintered funnel, washed with AcOEt and

80 °C. After work-up and purification (from CH₂Cl₂/ AcOEt 3:4 concentrated. The residue was treated with saturated aqueous

to AcOEt with 1% MeOH) compound 1h was obtained as red NaHCO₃and extracted with AcOEt. The organic extracts were

solid (60 mg, 74%). A final treatment with active coal gave 1h as washed with saturated brine and evaporated. The crude was

a pale yellow solid. M.p. = 158.0 – 160.0 °C (MeOH). R_f= 0.35 purified by chromatography. A 0.1 M solution of the Ugi product

(CH₂Cl₂/ AcOEt 3:4). δ_H(300 MHz, DMSO-d6, 90 °C) (Note: the 3 in MeCN under nitrogen atmosphere, was treated with

phenolic OH exchange with H₂O contained in the solvent giving PdCl₂(PPh₃)₂(0.025 equiv for allyl group) and ammonium

a broad signal around 4.90 ppm): 9.38 (1 H, s, NH), 9.30 (1 H, s, formate (2.2 equiv for allyl group) at 80 °C in a sealed flask (2-5

OH), 7.32 (2 H, d, J 8.8, H ortho to CH), 7.0-6.80 (2 H, broad h). Then, the crude was diluted with AcOEt, washed with

signal, H ortho to N), 6.86 (2 H, d, J 8.4, H ortho to NH), 6.67 (2 saturated aqueous NaHCO₃. After evaporation, the residue was

H, d, J 8.8, H meta to CH), 6.50 (4 H, d, J 8.4, H meta to N and to directly dissolved in 1:1 pyridine / acetic anhydride (0.1 M) and

NH), 6.03 (2 H, s, CH), 1.97 (2 H, q, J 7.4, CH₂CH₃), 0.92 (3 H, t, J stirred for 18 h at r.t. Then, the mixture was poured in 2 N HCl

7.5, CH₂CH₃). δ_C(75 MHz, DMSO-d6, 25 °C): 173.1, 168.8 (C=O), (final pH = 2) and extracted with CH₂Cl₂. The organic extracts

157.5, 157.0, 153.3, 130.7, 130.2, 124.5 (quat.), 131.7 (x2), were washed with saturated brine and evaporated, then the

131.2 (x2), 120.15 (x2), 115.0 (x2), 114.8 (x4) (ArCH), 63.6 (CHN), crude was purified by chromatography.

27.6 (CH₂), 9.4 (CH₃). IR: 

_max/cm^-13268, 3202, 3005, 1650, (R,S)-(E)-3-(4-Acetoxy-3-methoxyphenyl)-N-(1-(4-

1602, 1584, 1533, 1471, 1379, 1360, 1260, 1205, 1173, 1099, acetoxyphenyl)-2-(tert-butylamino)-2-oxoethyl)-N-

1043, 1001, 965, 845, 815, 631. m/z (ESI+): 407.1605 (M + H⁺). butylacrylamide 2d. Following the general procedure B, a

C₂₃H₂₃O₅N₂requires 407.1607. HPLC (see supplementary mixture of aldehyde 12 (176 mg, 1.08 mmol), n-butylamine (120

information) showed a purity of 98%.

(R,S)-(E)-N-(2-(tert-butylamino)-1-(4-hydroxyphenyl)-2-

oxoethyl)-3-(4-hydroxy-3-methoxyphenyl)-N-(4-

µL, 1.19 mmol), acid 13 (280 mg, 1.19 mmol), t-butyl isocyanide

(135 µL, 1.19 mmol) and 3 Å molecular sieves (50 mg) was

stirred for 3 days at r.t. After work-up and purification (PE /

hydroxyphenyl)acrylamide 1i. Following the general procedure AcOEt 75:25 + 1% EtOH) compound 14d was obtained pure as

A, a mixture of aldehyde 12 (94 mg, 0.58 mmol), 4- yellow oil (330 mg, 57%, 91% based on the recovery of

allyloxyaniline (95 mg, 0.64 mmol), acid 13 (150 mg, 0.64 mmol), unreacted aldehyde). Then a mixture of 14d (270 mg, 0.50

t-butyl isocyanide (72 µL, 0.64 mmol) and 3 Å molecular sieves mmol), PdCl₂(PPh₃)₂(18 mg, 0.0025 mmol) and ammonium

(29 mg) was stirred for 3 days at r.t. After work-up and formate (140 mg, 2.22 mmol) was stirred for 5 h at 80 °C. After

purification (PE / AcOEt 3:2) compound 14i was obtained pure work-up, the crude was treated with 1:1 pyridine / acetic

as yellow foam (117 mg, 33%). Then a mixture of 14i (88 mg, anhydride (3.7 mL) and stirred for 18 h at r.t. After work-up and

0.15 mmol), PdCl₂(PPh₃)₂(8 mg, 0.011 mmol) and ammonium purification (PE / AcOEt 7:3 + 3% EtOH) compound 2d was

formate (63 mg, 1.00 mmol) was stirred for 2 h at 80 °C. After obtained pure as white foam (226 mg, 76%). R_f= 0.28 (PE /

work-up and purification (the crude was triturated with AcOEt 7:3 + 3% EtOH). δ_H(300 MHz, CDCl₃, 25 °C): δ 7.72 (1 H, d,

14 | J. Name., 2012, 00, 1-3

This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins

Page 15 of 20

Organic & Biomolecular Chemistry

Please do not adjust margins

Organic and Biomolecular Chemistry

ARTICLE

J 15.3, ArCH=CH), 7.47 (2 H, d, J 8.4), 7.15-7.01 (5 H, m), 6.78 (1 brown foam (119 mg, 28%). Then a mixture of 14 g_V(_ie9_w4_Am_rticg_le,_O0_n._l1_in6_e

DOI: 10.1039/C7OB02182H

H, d, J 15.3, ArCH=CH), 6.04 (1 H, s, CH), 5.87 (1 H, s, NH), 3.85 mmol), PdCl₂(PPh₃)₂(9 mg, 0.01 mmol) and ammonium formate

(3 H, s, OCH₃), 3.57-3.32 (mc = 3.46) (2 H, m, CH₂N), 2.32, 2.31 (67 mg, 1.06 mmol) was stirred for 4 h at 80 °C. After work-up,

(2 x 3 H, 2 s, CH₃CO), 1.55-1.39 (1 H, m, CHH), 1.37 (9 H, s, the crude was treated with 1:1 pyridine / acetic anhydride (3

(CH₃)₃C), 1.22-0.95 (3 H, m, CH₂and CHH), 0.79 (3 H, t, J 7.2, mL) and stirred for 18 h at r.t. After work-up and purification (PE

CH₃CH₂). δ_H(75 MHz, CDCl₃, 25 °C): 169.3, 168.9 (x2), 166.9 / AcOEt 1:1) pure compound 2g was obtained as white foam (55

(C=O), 151.3, 150.6, 140.9, 134.3, 133.4 (quat.), 142.8 mg, 59%). R_f= 0.72 (PE / AcOEt 3:7). δ_H(300 MHz, CDCl₃, 25 °C):

(ArCH=CH), 130.5 (x2), 123.2, 121.9 (x2), 120.4, 111.6 (ArCH), 8.43 (1 H, s, NH), 7.48 (2 H, d, J 8.8), 7.28 (4 H, d, J 8.1), 7.25-

118.0 (ArCH=CH), 61.5 (CH), 58.8 (OCH₃), 51.7 (C(CH₃), 46.1 7.10 (3 H, m), 7.02-6.93 (6 H, m), 6.78 (2 H, d, J 8.5), 6.42 (1 H,

(NCH₂), 32.6 (NCH₂CH₂), 28.6 (C(CH₃)₃), 21.1, 20.7 (CH₃CO), 20.0 s, CH), 2.26 (6 H, s, CH₃CO), 2.19 (3 H, s, CH₃CO). δ_H(75 MHz,

(CH₂CH₃), 13.5 (CH₂CH₃). IR: 

_max/cm^-13676, 3295, 3056, 2965, CDCl₃, 25 °C): δ 171.6, 169.5, 169.1, 168.7, 167.9 (C=O), 150.9,

2930, 2877, 2856, 1768, 1742, 1684, 1640, 1608, 1582, 1542, 149.5, 146.9, 138.0, 135.4, 135.3, 129.9 (quat.), 131.51 (x2),

1508, 1486, 1473, 1454, 1431, 1407, 1392, 1364, 1349, 1311, 131.43 (x2), 131.37, 128.6 (x2), 127.8 (x2), 121.9 (x4), 121.5 (x2),

1302, 1288, 1268, 1259, 1246, 1208, 1194, 1166, 1123, 1066, 120.9 (x2) (ArCH), 66.2 (CH), 21.1 (CH₃CO). IR: 

_max/cm^-13275,

1045, 1032, 1012, 979, 951, 937, 918, 906, 889, 865, 842, 815, 3054, 2980, 1743, 1691, 1615, 1594, 1533, 1551, 1488, 1463,

801, 792, 753, 737, 731, 709, 688, 633, 620. m/z (ESI+): 1401, 1333, 1301, 1198, 1133, 1101, 1045, 1007, 965, 841, 761,

539.2758 (M + H⁺). C₃₀H₃₉O₇N₂requires 539.2757.

(R,S)-(E)-N-(1-(4-Acetoxyphenyl)-2-(tert-butylamino)-2-

oxoethyl)-3-(3,4-diacetoxyphenyl)-N-benzylacrylamide

732, 679, 630, 603. m/z (ESI+): 581.1914 (M + H⁺). C₃₃H₂₉O₈N₂

requires 581.19244.

(R,S)-4-Acetoxy-N-(1-(4-Acetoxyphenyl)-2-((4-

2f

.

Following the general procedure B, a mixture of aldehyde 12 acetoxyphenyl)amino)-2-oxoethyl)-N-phenylbenzamide

2j.

(596 mg, 3.70 mmol), benzylamine (445 µL, 4.07 mmol), (E)-3- Following the general procedure B, a mixture of aldehyde 12

(3,4-bis(allyloxy)phenyl)acrylic acid (1.06 g, 4.07 mmol), t-butyl (122 mg, 0.75 mmol), aniline (74 µL, 0.82 mmol), 4-

isocyanide (460 µL, 4.07 mmol) and 3 Å molecular sieves (200 (allyloxy)benzoic acid (146 mg, 0.82 mmol), 4-allyloxyphenyl

mg) was stirred for 3 days at r.t. After work-up and purification isocyanide (130 mg, 0.82 mmol) and 3 Å molecular sieves (70

(PE / AcOEt 2:1) compound 14f was obtained pure as white mg) was stirred for 3 days at r.t. After usual work-up, the crude

foam (1.59 g, 73%). Then a mixture of 14f (710 mg, 1.20 mmol), was diluted with AcOEt, washed with HCl 1 N to remove the

PdCl₂(PPh₃)₂(70 mg, 0.1 mmol) and ammonium formate (500 excess of amine. Then, the crude was purified (PE / AcOEt 7:3)

mg, 7.90 mmol) was stirred for 2 h at 80 °C. After work-up, the obtaining compound 14j as yellow oil (74 mg, 17%). Then a

crude was treated with 1:1 pyridine / acetic anhydride (8 mL) mixture of 14j (74 mg, 0.13 mmol), PdCl₂(PPh₃)₂(7 mg, 0.01

and stirred for 18 h at r.t. After work-up and purification (the mmol) and ammonium formate (54 mg, 0.86 mmol) was stirred

crude was triturated with AcOEt and the mother liquor was for 3 h at 80 °C. After work-up, the crude was treated with 1:1

purified by chromatography PE / Et₂O 1:2 + 2% EtOH) pure pyridine / acetic anhydride (3 mL) and stirred for 18 h at r.t.

compound 2f was obtained as white solid (602 mg, 83%). M.p. After work-up and purification (PE / AcOEt from 3:2 to 1:1) pure

= 189.0 – 190.0 °C (CH₂Cl₂). R_f= 0.70 (CH₂Cl₂/ MeOH 15:1). compound 2j was obtained as white foam (48 mg, 64%). R_f=

δ_H(300 MHz, CDCl₃, 25 °C): δ 7.70 (1 H, d, J 15.4, ArCH=CH), 7.38 0.50 (PE / AcOEt 3:7). δ_H(300 MHz, CDCl₃, 25 °C): 8.05 (1 H, s,

(2 H, d, J 8.4), 7.25-7.10 (6 H, m), 7.02-6.92 (4 H, m), 6.66 (1 H. NH), 7.55 (2 H, d, J 8.9), 7.36 (2 H, d, J 8.7), 7.35 (2 H, d, J 8.4),

d, J 15.4, ArCH=CH), 6.06 (1 H, s, CH), 5.67 (1 H, s, NH), 4.90 (1 7.12-6.96 (9 H, m), 6.89 (2 H, d, J 8.7), 6.28 (1 H, s, CH), 2.28 (6

H, d, J 17.5, CHPh), 4.66 (1 H, d, J 17.5, CHPh), 2.27 (3 H, s, H, s, CH₃CO), 2.22 (3 H, s, CH₃CO). δ_C(75 MHz, CDCl₃, 25 °C):

CH₃CO), 2.26 (6 H, s, CH₃CO), 1.35 (9 H, s, (CH₃)₃C). δ_C(75 MHz, 170.5, 169.5, 169.1, 168.7, 167.8 (C=O), 151.5, 150.8, 146.8,

CDCl₃, 25 °C): 169.1, 168.6, 168.0 (x2), 167.6 (C=O), 150.6, 143.0, 140.6, 135.5, 132.9, 131.4 (quat.), 131.3 (x2), 130.4 (x2), 130.2

142.2, 137.9, 134.0, 132.6 (quat.), 141.9 (ArCH=CH), 130.8 (x2), (x2), 128.6 (x2), 127.6, 121.9 (x2), 121.7 (x2), 120.9 (x2), 120.8

128.5 (x2), 127.0, 126.1 (x3), 123.7, 122.6, 121.7 (x2) (ArCH), (x2) (ArCH), 66.6 (CH), 21.1 (CH₃CO). IR:

119.2 (ArCH=CH), 62.0 (CH), 51.7 (C(CH₃), 49.6 (NCH₂), 28.6 2988, 1756, 1697, 1621, 1595, 1546, 1505, 1494, 1453, 1409,



_max/cm^-13282, 3070,

(C(CH₃)₃), 21.1, 20.6, 20.5 (CH₃CO). IR: 

_max/cm^-13295, 3071, 1367, 1310, 1187, 1163, 1106, 1075, 1046, 1014, 966, 909, 846,

2939, 1758, 1694, 1646, 1602, 1546, 1505, 1451, 1408, 1368, 757, 737, 700, 675, 634, 611. m/z (ESI+): 581.1929 (M + H⁺).

1302, 1258, 1185, 1162, 1122, 1013, 979, 953, 909, 832, 793, C₃₃H₂₉O₈N₂requires 581.1924.

730, 692, 638. m/z (ESI-): 599.2381 (M - H⁺). C₃₄H₃₅O₈N₂requires (R,S)-4-Acetoxy-N-(4-acetoxyphenyl)-N-(1-(4-

599.2393.

(pivaloyloxy)phenyl)-2-((4-acetoxyphenyl)amino)-2-

(R,S)-N-(4-Acetoxyphenyl)-N-(1-(4-Acetoxyphenyl)-2-((4-

oxoethyl)benzamide 2k. Following the general procedure B, a

acetoxyphenyl)amino)-2-oxoethyl)benzamide 2g

. Following mixture of 4-pivaloyloxybenzaldehyde (203 mg, 1.00 mmol), 4-

the general procedure B, a mixture of aldehyde 12 (122 mg, 0.75 allyloxyaniline (153 µL, 1.11 mmol), 4-allyloxybenzoic acid (200

mmol), 4-allyloxyaniline (114 µL, 0.82 mmol), benzoic acid (100 mg, 1.11 mmol), and 2,6-dimethylphenyl isocyanide (144 mg,

mg, 0.82 mmol), 4-allyloxyphenyl isocyanide (130 mg, 0.82 1.11 mmol) and 3 Å molecular sieves (50 mg) was stirred for 3

mmol) and 3 Å molecular sieves (70 mg) was stirred for 3 days days at r.t. After work-up and purification (PE / AcOEt 7:3)

at r.t. After usual work-up, the crude was diluted with AcOEt, compound 15 was obtained pure as off-white foam (198 mg,

washed with HCl 1 N to remove the excess of amine. Then, the 31%). Then a mixture of 15 (168 mg, 0.26 mmol), PdCl₂(PPh₃)₂

crude was purified (PE / AcOEt 3:2) obtaining compound 14g as (9 mg, 0.013 mmol) and ammonium formate (72 mg, 1.14

J. Name., 2013, 00, 1-3 | 15

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 16 of 20

ARTICLE

Organic and Biomolecular Chemistry

mmol) was stirred for 5 h at 80 °C. After work-up, the crude was acetic anhydride (9 mL) and stirred for 18 h at r.t. After work-up

View Article Online

treated with 1:1 pyridine / acetic anhydride (2.6 mL) and stirred and purification (PE / AcOEt from 1:3 to 1:4) compound

^{DOI: 10.1039/C7O}2^B0m²¹w⁸²a^Hs

for 18 h at r.t. After work-up and purification (PE / Et₂O 1:3 + 1% obtained pure as white foam (188 mg, 78%). R_f= 0.32 (PE /

EtOH) compound 2d was obtained pure as white foam (117 mg, AcOEt 1:6). δ_H(300 MHz, CDCl₃, 50 °C): 7.68 (1 H, d, J 15.3,

69%). R_f= 0.15 (PE / Et₂O 1:3 + 1% EtOH). δ_H(300 MHz, CDCl₃, 25 ArCH=CH), 7.56-7.45 (2 H, m), 7.13 (2 H, d, J 8.4), 7.11-6.89 (7 H,

°C): δ 7.42 (2 H, d, J 8.2), 7.42 (2 H, d, J 8.4), 7.26 (1 H, s, NH), m), 6.74 (1 H, d, J 15.3, ArCH=CH), 6.21 (1 H, broad d, J 4.8, NH),

7.20-6.98 (7 H, m), 6.90 (2 H, d, J 8.3), 6.82 (2 H, d, J 8.5), 6.28 6.12 (1 H, s, CH), 3.84 (3 H, s, OCH₃), 3.80-3.55 (2 H, broad m,

(1 H, s, CH), 2.23 (12 H, s, CH₃CO and CH₃Ar), 1.35 (9 H, s, CH₂N), 2.87-2.73 (1 H, m, ArCHH), 2.83 (3 H, d, J 4.8, CH₃NH),

C(CH₃)₃). δ_H(75 MHz, CDCl₃, 25 °C): 176.8, 170.3, 168.7 (x2), 2.45-2.17 (1 H, m, ArCH₂), 2.30, 2.29, 2.24 (3 x 3 H, 3 s, CH₃CO).

167.9 (C=O), 151.5 (x2), 149.6, 138.4, 135.5 (x2), 133.4, 133.0, δ_H(75 MHz, CDCl₃, 25 °C): 170.0, 169.2, 169.0, 168.6, 167.2

131.6 (quat.), 131.4 (x4), 130.0 (x2), 128.2 (x2), 127.4, 121.9 (C=O), 151.5, 151.0, 149.5, 141.3, 135.8, 134.1, 132.9 (quat.),

(x2), 121.6 (x2), 120.9 (x2) (ArCH), 66.3 (CH), 39.1 (C(CH₃)₃), 27.1 143.1 (ArCH=CH), 130.7 (x2), 129.5 (x2), 123.3, 122.1 (x2), 121.7

(ArCH₃), 21.1, 18.6 (CH₃CO). IR:

2874, 1752, 1643, 1603, 1503, 1479, 1418, 1367, 1279, 1263, (OCH₃), 48.0 (NCH₂), 36.4 (ArCH₂), 26.4 (CH₃NH), 21.0 (x2), 20.5

1189, 1164, 1107, 1015, 945, 910, 850, 803, 765, 703, 678, 626. (CH₃CO). IR:

_max/cm^-13311, 2941, 2249, 1759, 1674, 1647,



_max/cm^-13269, 3046, 2970, (x2), 120.6, 111.8 (ArCH), 117.8 (ArCH=CH), 61.6 (CH), 56.0



m/z (ESI+): 651.2713 (M + H⁺). C₃₈H₃₉O₈N₂requires 651.2706.

(R,S)-(E)-N-(3-Acetoxybenzyl)-N-(1-(4-acetoxyphenyl)-2-((4-

acetoxyphenyl)amino)-2-oxoethyl)-3-(4-acetoxy-3-

1601, 1506, 1466, 1450, 1416, 1369, 1262, 1190, 1153, 1121,

1032, 1013, 979, 908, 829, 726, 646, 623. m/z (ESI+): 603.2352

(M + H⁺). C₃₃H₃₅O₉N₂requires 603.2343.

methoxyphenyl)acrylamide 2l. Following the general (R,S)-(E)-3-(4-Acetoxy-3-methoxyphenyl)-N-(1-(4-acetoxy-3-

procedure B, a mixture of aldehyde 12 (404 mg, 2.49 mmol), 3- methoxyphenyl)-2-(tert-butylamino)-2-oxoethyl)-N-

allyloxybenzylamine (406 mg, 2.49 mmol), acid 13 (530 mg, 2.26 benzylacrylamide 2n. Following the general procedure B, a

mmol), 4-allyloxyphenyl isocyanide (397 mg, 2.49 mmol) and 3 mixture of 4-allyloxy-3-methoxybenzaldehyde (140 mg, 0.70

Å molecular sieves (250 mg) was stirred for 3 days at r.t. After mmol), benzylamine (84 µL, 0.77 mmol), acid 13 (180 mg, 0.77

work-up and purification (PE / AcOEt from 2:1 to 1:1) compound mmol), t-butyl isocyanide (87 µL, 0.77 mmol) and 3 Å molecular

14l was obtained as yellow foam (1.19 g, 75%). Then a mixture sieves (35 mg) was stirred for 3 days at r.t. After work-up and

of 14l (200 mg, 0.29 mmol), PdCl₂(PPh₃)₂(16 mg, 0.02 mmol) purification (PE / AcOEt 6:4) compound 14n was obtained pure

and ammonium formate (161 mg, 2.56 mmol) was stirred for 4 as pale yellow foam (249 mg, 63%). Then a mixture of 14n (249

h at 80 °C. After work-up, the crude was treated with 1:1 mg, 0.42 mmol), PdCl₂(PPh₃)₂(15 mg, 0.021 mmol) and

pyridine / acetic anhydride (4 mL) and stirred for 18 h at r.t. ammonium formate (117 mg, 1.85 mmol) was stirred for 5 h at

After work-up and purification (PE / AcOEt 3:4) compound 2l 80 °C. After work-up, the crude was treated with 1:1 pyridine /

was obtained pure as white foam (118 mg, 58%). R_f= 0.80 (PE / acetic anhydride (4.2 mL) and stirred for 18 h at r.t. After work-

AcOEt 1:6). δ_H(300 MHz, CDCl₃, 25 °C): 8.50 (1 H, s, NH), 7.67 (1 up and purification (PE / AcOEt 6:4 + 3% EtOH) compound 2n

H, d, J 15.3, ArCH=CH), 7.48 (2 H, d, J 8.7), 7.42 (2 H, d, J 8.4), was obtained pure as white foam (144 mg, 36%). R_f= 0.26 (PE /

7.20 (1 H, t, J 7.9), 7.03-6.80 (10 H, m), 6.67 (1 H, d, J 15.3, AcOEt 6:4 + 3% EtOH). δ_H(300 MHz, CDCl₃, 25 °C): 7.71 (1 H, d, J

ArCH=CH), 6.30 (1 H, s, CH), 4.93 and 4.69 (2 H, AB syst., J 17.9, 15.3, ArCH=CH), 7.45-7.10 (4 H, m), 7.10-6.90 (6 H, m), 6.83 (1

CH₂Ar), 3.76 (3 H, s, OCH₃), 2.30, 2.26, 2.24 (4 x 3 H, 4 s, CH₃CO). H, s), 6.65 (1 H, d, J 15.3, ArCH=CH), 6.12 (1 H, s, CH), 5.76 (1 H,

δ_C(75 MHz, CDCl₃, 25 °C): 169.5, 169.2, 169.1, 168.8, 168.3, s, NH), 4.92, 4.68 (2 H, AB syst., J 17.9, CH₂Ph), 3.74, 3.67 (2 x 3

167.9 (C=O), 151.2, 150.9 (x2), 146.9, 141.1, 139.5, 135.4, 133.7, H, 2 s, OCH₃), 2.29, 2.28 (2 x 3 H, 2 s, CH₃CO), 1.37 (9 H, s,

131.5, 144.2 (ArCH=CH), 131.0 (x2), 129.6, 123.6, 123.1, 122.0 C(CH₃)₃). δ_C(75 MHz, CDCl₃, 25 °C): δ 168.8 (x2), 168.0 (x2) (C=O),

(x2), 121.9 (x2), 120.9 (x3), 120.4, 119.6, 111.5 (ArCH), 117.6 151.1, 151.0, 140.9, 139.8, 138.4, 134.1, 133.9 (quat.), 143.0

(ArCH=CH), 63.0 (CH), 55.9 (OCH₃), 49.6 (ArCH₂), 21.10 (x2), (ArCH=CH), 128.5 (x2), 127.0, 126.2 (x2), 123.0, 122.8, 122.0,

21.08, 20.6 (CH₃CO). IR:

1647, 1602, 1545, 1505, 1452, 1408, 1368, 1302, 1258, 1185, (OCH₃), 51.8 (C(CH₃)₃), 49.6 (NCH₂), 28.6 (C(CH₃)₃), 20.6 (CH₃CO).



_max/cm^-13285, 3072, 2940, 1758, 1694, 120.8, 114.1, 111.3 (ArCH), 118.6 (ArCH=CH), 62.2 (CH), 55.8

1161, 1122, 1013, 978, 955, 908, 831, 792, 730, 691, 637. m/z IR:

(ESI+): 709.2405 (M + H⁺). C₃₉H₃₇O₁₁N₂requires 709.2397.

(R,S)-(E)-N-(2-(4-Acetoxyphenyl)ethyl)-N-(1-(4-



_max/cm^-13279, 3064, 2939, 1761, 1735, 1688, 1646, 1601,

1547, 1507, 1454, 1414, 1368, 1298, 1213, 1191, 1156, 1121,

1031, 1012, 976, 953, 909, 829, 723, 696, 635, 603. m/z (ESI+):

603.2697 (M + H⁺). C₃₄H₃₉O₈N₂requires 603.2706.

acetoxyphenyl)-2-(methylamino)-2-oxoethyl)-3-(4-acetoxy-3-

methoxyphenyl)acrylamide 2m. Following the general (R,S)-(E)-3-(4-Acetoxy-3-methoxyphenyl)-N-(1-(4-

procedure B, a mixture of aldehyde 12 (135 mg, 0.83 mmol), 4- acetoxyphenyl)-2-(methylamino)-2-oxoethyl)-N-

allyloxyphenethylamine (148 mg, 0.83 mmol), acid 13 (176 mg, benzylacrylamide 2o. Following the general procedure B, a

0.75 mmol), methyl isocyanide (74

L, 1.23 mmol) and 3 Å mixture of 4-allyloxybenzaldehyde (150 mg, 0.93 mmol),

molecular sieves (50 mg) was stirred for 3 days at r.t. After work- benzylamine (111 µL, 1.02 mmol), acid 13 (238 mg, 1.02 mmol),

up and purification (PE / AcOEt 1:2) compound 14m was methyl isocyanide (61 µL, 1.02 mmol) and 3 Å molecular sieves

obtained as yellow foam (263 mg, 59%). Then a mixture of 14m (47 mg) was stirred for 3 days at r.t. After work-up and

(237 mg, 0.40 mmol), PdCl₂(PPh₃)₂(23 mg, 0.03 mmol) and purification (PE / AcOEt 4:6) compound 14o was obtained pure

ammonium formate (170 mg, 2.70 mmol) was stirred for 4 h at as pale yellow foam (246 mg, 51%). Then a mixture of 14o (229

80 °C. After work-up, the crude was treated with 1:1 pyridine / mg, 0.43 mmol), PdCl₂(PPh₃)₂(15 mg, 0.022 mmol) and

16 | J. Name., 2012, 00, 1-3

Please do not adjust margins

Page 17 of 20

Organic & Biomolecular Chemistry

Please do not adjust margins

Organic and Biomolecular Chemistry

ARTICLE

ammonium formate (120 mg, 1.91 mmol) was stirred for 5 h at mg, 0.83 mmol), acid 13 (176 mg, 0.75 mmol), methyl

View Article Online

DOI: 10.1039/C7OB02182H

80 °C. After work-up, the crude was treated with 1:1 pyridine / isocyanide (49 L, 0.83 mmol) and 3 Å molecular sieves (50 mg)

acetic anhydride (4.3 mL) and stirred for 18 h at r.t. After work- was stirred for 3 days at r.t. After work-up and purification (PE

up and purification (PE / AcOEt 4:6 + 3% EtOH) compound 2o / AcOEt 3:7) compound 14q was obtained as white foam (313

was obtained pure as white foam (168 mg, 74%). R_f= 0.40 (PE / mg, 72%). Then a mixture of 14q (97 mg, 0.17 mmol),

AcOEt 4:6 + 3% EtOH). δ_H(300 MHz, CDCl₃, 25 °C): 7.70 (1 H, d, J PdCl₂(PPh₃)₂(10 mg, 0.014 mmol) and ammonium formate (71

15.3, ArCH=CH), 7.43 (2 H, d, J 8.4), 7.27-7.13 (3 H, m), 7.09 (2 mg, 1.12 mmol) was stirred for 3 h at 80 °C. After work-up, the

H, broad d, J 7.2), 7.05-6.93 (4 H, m), 6.84 (1 H, s), 6.63 (1 H, d, crude was treated with 1:1 pyridine / acetic anhydride (2.5 mL)

J 15.3, ArCH=CH), 5.99 (1 H, s, CH), 5.94 (1 H, s, NH), 4.89, 4.66 and stirred for 18 h at r.t. After work-up and purification (PE /

(2 H, AB syst., J 17.8, CH₂Ph), 3.75 (3 H, s, OCH₃), 2.84 (3 H, d, J AcOEt 1:6) compound 2q was obtained pure as white foam (73

4.8, CH₃NH), 2.29, 2.28 (2 x 3 H, 2 s, CH₃CO). δ_C(75 MHz, CDCl₃, mg, 75%). R_f= 0.49 (PE / AcOEt 1:6). δ_H(300 MHz, CDCl₃, 25 °C):

25 °C): 169.9, 169.2, 168.8, 168.0 (C=O), 151.2, 150.7, 141.0, 7.72 (1 H, d, J 15.3, ArCH=CH), 7.40 (2 H, d, J 8.1), 7.21 (1 H, t, J

137.7, 134.0, 132.5 (quat.), 143.3 (ArCH=CH), 130.9 (x2), 128.6 7.8), 7.03-6.82 (8 H, m), 6.63 (1 H, d, J 15.3, ArCH=CH), 5.97 (1

(x2), 127.2, 126.2 (x2), 123.1, 121.9 (x2), 120.8, 111.3 (ArCH), H, s, CH), 5.94 (1 H, broad s, NH), 4.89, 4.65 (2 H, AB syst., J 17.9,

118.3 (ArCH=CH), 62.5 (CH), 55.8 (OCH₃), 50.2 (NCH₂), 26.5 CH₂Ar), 3.77 (3 H, s, OCH₃), 2.83 (3 H, d, J 4.6, CH₃N), 2.29, 2.27,

(NCH₃), 21.1, 20.6 (CH₃CO). IR: 

_max/cm^-13302, 3065, 2940, 2.26 (3 x 3 H, 3 s, CH₃CO). δ_C(75 MHz, CDCl₃, 25 °C): 169.9,

1760, 1674, 1647, 1601, 1506, 1453, 1407, 1368, 1300, 1257, 169.21, 169.16, 168.8, 168.0 (C=O), 151.2, 150.9, 150.8, 141.0,

1189, 1155, 1121, 1080, 1030, 1012, 976, 957, 907, 844, 829, 139.6, 133.9, 132.2 (quat.), 143.8 (ArCH=CH), 131.0 (x2), 129.6,

724, 697, 676, 635. m/z (ESI+): 531.2137 (M + H⁺). C₃₀H₃₁O₇N₂123.6, 123.1, 121.9 (x2), 120.9, 120.4, 119.6, 111.4 (ArCH),

requires 531.2131.

(R,S)-(E)- N-(2-((4-(2-(2-Acetoxyethoxy)ethoxy)phenyl)amino)- (CH₃N), 21.1 (x2), 20.6 (CH₃CO). IR:

1-(4-acetoxyphenyl)-2-oxoethyl)-3-(4-acetoxy-3- 2940, 1760, 1676, 1648, 1602, 1506, 1438, 1414, 1368, 1302,

117.9 (ArCH=CH), 62.5 (CH), 55.9 (OCH₃), 49.7 (NCH₂), 26.4



_max/cm^-13302, 3072, 2967,

methoxyphenyl)-N-benzylacrylamide 2p. Following the general 1258, 1190, 1156, 1120, 1014, 975, 909, 829, 794, 751, 722, 694,

procedure B, a mixture of 4-allyloxybenzaldehyde (146 mg, 0.90 635. m/z (ESI+): 589.2200 (M + H⁺). C₃₂H₃₃O₉N₂requires

mmol), benzylamine (106 µL, 0.99 mmol), acid 13 (231 mg, 0.99 589.2186.

mmol), N-(4-(2-(2-(allyloxy)ethoxy)ethoxy)phenyl) isocyanide General procedure for the preparation of polyphenols

(244 mg, 0.99 mmol) and 3 Å molecular sieves (45 mg) was 1d,f,g,h,j,k,l,m,n,o,p,q from the corresponding acetylated

stirred for 3 days at r.t. After work-up and purification (PE / derivatives 2. The peracylated Ugi product was treated with 0.2

AcOEt 1:1) compound 14p was obtained pure as pale yellow M MeONa in MeOH (freshly prepared by adding Na to dry

foam (449 mg, 71%). Then a mixture of 14p (449 mg, 0.61 MeOH) under N₂atmosphere. After stirring for 1 h (18 h for

mmol), PdCl₂(PPh₃)₂(32 mg, 0.046 mmol) and ammonium compound 15) at r.t., the mixture was treated with previously

formate (254 mg, 4.03 mmol) was stirred for 5 h at 80 °C. After washed Amberlyst 15 acid resin until pH = 4. The resin was

work-up, the crude was treated with 1:1 pyridine / acetic filtered off and the solution evaporated to dryness (with

anhydride (6.1 mL) and stirred for 18 h at r.t. After work-up and exception of compound 1n). The resulting polyphenols were not

1

purification (PE / AcOEt 1:1 + 3% EtOH) compound 2p was fully characterized, but only examined at H NMR and HPLC in

obtained pure as white foam (271 mg, 60%). R_f= 0.40 (PE / order to establish their degree of purity.

AcOEt 4:6 + 3% EtOH). δ_H(300 MHz, CDCl₃, 25 °C): δ 7.90 (1 H, s, (R,S)-(E)-N-Butyl-N-(2-(tert-butylamino)-1-(4-hydroxyphenyl)-

NH), 7.71 (1 H, d, J 15.2, ArCH=CH), 7.49 (2 H, d, J 8.2), 7.39 (2 2-oxoethyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide 1d

.

H, d, J 8.7), 7.30-7.07 (5 H, m), 7.03 (2 H, d, J 8.3), 6.96 (2 H, s), δ_H(300 MHz, DMSO-d6, 90 °C) (Note: the 2 phenolic OH

6.90-6.81 (3 H, m), 6.67 (1 H, d, J 15.3, ArCH=CH), 6.19 (1 H, s, exchange with H₂O contained in the solvent giving a broad

CH), 4.92, 4.72 (2 H, AB syst., J 17.6, CH₂Ph), 4.25 (2 H, dd, J 4.5, signal around 9 ppm): 7.42 (1 H, d, J 15.2, ArCH=CH), 7.35 (1 H,

5.0, OCH₂), 4.10 (2 H, broad t, J 4.5, CH₂O), 3.83 (2 H, broad t, J s, NH), 7.17 (1 H, d, J 1.8), 7.14 (2 H, d, J 8.7), 7.06 (1 H, dd, J 8.2,

4.5, CH₂O), 3.76 (2 H, dd, J 4.5, 5.0, OCH₂), 3.75 (3 H, s, OCH₃), 1.8), 6.82 (1 H, d, J 15.2, ArCH=CH), 6.81 (1 H, d, J 8.2), 6.79 (2

2.29, 2.28, 2.07 (3 x 3 H, 3 s, CH₃CO). δ_C(75 MHz, CDCl₃, 25 °C): H, d, J 8.7), 5.87 (1 H, s, CH), 3.83 (3 H, s, OCH₃), 3.50-3.25 (mc =

δ 171.1, 169.1, 168.3, 167.6 (C=O), 155.6, 151.2, 150.8, 141.0, 3.38) (2 H, m, CH₂N), 1.50-1.30 (1 H, m, CHH), 1.29 (9 H, s,

137.7, 133.9, 135.5, 132.0 (quat.), 143.6 (ArCH=CH), 130.8 (x2), (CH₃)₃C), 1.20-0.95 (3 H, m, CH₂and CHH), 0.74 (3 H, t, J 7.2,

128.7 (x2), 127.3, 126.3 (x2), 123.1, 122.0 (x2), 121.8 (x2), 120.8, CH₃CH₂). HPLC (see supplementary information) showed a

114.9 (x2), 111.4 (ArCH), 118.1 (ArCH=CH), 69.7, 69.3, 67.7, 63.6 purity of 92%.

(CH₂O), 63.0 (CH), 55.8 (OCH₃), 50.2 (NCH₂), 21.1, 21.0, 20.6 (R,S)-(E)-N-Benzyl-N-(2-(tert-butylamino)-1-(4-

(CH₃CO). IR: 

_max/cm^-13278, 3065, 2940, 1760, 1735, 1688, hydroxyphenyl)-2-oxoethyl)-3-(3,4-

1647, 1601, 1546, 1507, 1454, 1414, 1368, 1299, 1191, 1157, dihydroxyphenyl)acrylamide 1f. δ_H(300 MHz, DMSO-d6, 70 °C)

1121, 1031, 1012, 975, 953, 909, 829, 723, 697, 636, 604. m/z (Note: the 2 phenolic OH exchange with H₂O contained in the

(ESI+): 739.2875 (M + H⁺). C₄₁H₄₃O₁₁N₂requires 739.2867.

solvent giving a very broad signal around 9 ppm): 7.53 (1 H, s,

(R,S)-(E)-N-(3-acetoxybenzyl)-3-(4-acetoxy-3-methoxyphenyl)- NH), 7.35 (1 H, d, J 15.2, ArCH=CH), 7.20-7.00 (6 H, m, ArCH),

N-(1-(4-acetoxyphenyl)-2-(methylamino)-2-oxoethyl)- 6.95-6.75 (2 H, m, ArCH), 6.73-6.62 (4 H, m, ArCH=CH and ArCH),

acrylamide 2q. Following the general procedure B, a mixture of 6.01 (1 H, s, CH), 4.85 (1 H, d, J 16.9, CHHPh), 4.55 (1 H, d, J 16.9,

aldehyde 12 (135 mg, 0.83 mmol), 3-allyloxybenzylamine (135

J. Name., 2013, 00, 1-3 | 17

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 18 of 20

ARTICLE

Organic and Biomolecular Chemistry

CHHPh), 1.24 (9 H, s, (CH₃)₃C). HPLC (see supplementary H, s, CH), 4.87, 4.55 (2 H, AB syst., J 16.7, PhCH₂), 3.78 (3 H, s,

View Article Online

DOI: 10.1039/C7OB02182H

information) showed a purity of 97%.

(R,S)-N-(4-Hydroxyphenyl)-N-(1-(4-hydroxyphenyl)-2-((4-

OCH₃), 3.63 (3 H, s, OCH₃), 1.27 (9 H, s, (CH₃)₃C). HPLC (see

supplementary information) showed a purity of 99%.

hydroxyphenyl)amino)-2-oxoethyl)benzamide 1g

.

δ_H(300 (R,S)-(E)-N-Benzyl-3-(4-hydroxy-3-methoxyphenyl)-N-(1-(4-

MHz, DMSO-d6, 70 °C) (Note: the 3 phenolic OH exchange with hydroxyphenyl)-2-(methylamino)-2-oxoethyl)-acrylamide 1o

.

H₂O contained in the solvent giving a very broad signal not δ_H(300 MHz, DMSO-d6, 90 °C): 9.09, 9.00 (2 x 1 H, 2 broad s,

visible in the spectrum): 9.69 (1 H, s, NH), 7.39 (2 H, d, J 8.8), OH), 7.73 (1 H, s, NH), 7.40 (1 H, d, J 15.3, ArCH=CH₂), 7.21-7.02

7.25-7.12 (5 H, m), 6.96 (2 H, d, J 8.5), 6.81 (2 H, broad d, J 7.2), (6 H, m), 6.96 (1 H, s), 6.90 (1 H, d, J 8.1), 6.75 (2 H, d, J 8.4), 6.67

6.70 (2 H, d, J 9.0), 6.57 (2 H, d, J 8.7), 6.33 (2 H, d, J 8.9), 6.21 (1 (2 H, d, J 8.7), 6.66 (1 H, broad signal, ArCH=CH), 6.04 (1 H, s,

H, s, CH). HPLC (see supplementary information) showed a CH), 4.85, 4.62 (2 H, AB syst., J 17.1, CH₂Ph), 3.77 (3 H, s, OCH₃),

purity of 99%.

2.63 (3 H, d, J 4.6, CH₃NH). HPLC (see supplementary

information) showed a purity of 96%.

(R,S)-(E)-N-Benzyl-N-(2-((4-(2-(2-

(R,S)-4-Hydroxy-N-(1-(4-hydroxyphenyl)-2-((4-

hydroxyphenyl)amino)-2-oxoethyl)-N-phenylbenzamide 1j

.

δ_H(300 MHz, DMSO-d6, 30 °C) (Note: the 3 phenolic OH hydroxyethoxy)ethoxy)phenyl)amino)-1-(4-hydroxyphenyl)-2-

exchange with H₂O contained in the solvent giving a very broad oxoethyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide 1p.

signal not visible in the spectrum): δ 9.91 (1 H, s, NH), 7.40 (2 H, δ_H(300 MHz, DMSO-d6, 90 °C): 9.80 (1 H, s, NH), 9.15, 9.01 (2 x

d, J 8.9), 7.04 (2 H, d, J 8.7), 6.99 (5 H, broad s), 6.93 (2 H, d, J 1 H, 2 broad s, OH), 7.46 (2 H, d, J 9.0), 7.43 (1 H, d, J 15.0,

8.4), 6.69 (2 H, d, J 8.9), 6.53 (2 H, d, J 8.4), 6.49 (2 H, d, J 8.7), ArCH=CH), 7.22-7.06 (4 H, m), 6.94 (1 H, broad d, J 15.0,

6.22 (1 H, s, CH). HPLC (see supplementary information) showed ArCH=CH), 6.88 (2 H, d, J 9.0), 6.75 (1 H, d, J 8.1), 6.69 (2 H, d, J

a purity of 100%.

(R,S)-4-Hydroxy-N-(4-hydroxyphenyl)-N-(1-(4-

(hydroxy)phenyl)-2-((4-hydroxyphenyl)amino)-2-

8.4), 6.21 (1 H, s, CH), 4.90, 4.63 (2 H, AB syst., J 17.1, CH₂Ph),

4.08 (2 H, t, J 5.0, OCH₂), 3.75 (2 H, t, J 4.8, CH₂O), 3.75 (3 H, s,

OCH₃), 3.58-3.48 (4 H, m, CH₂O). HPLC (see supplementary

oxoethyl)benzamide 1k. δ_H(300 MHz, DMSO-d6, 70 °C): δ 9.39, information) showed a purity of 97.5%.

9.14, 9.03 (3 x 1 H, 3 broad s, OH), 9.21 (1 H, s, NH), 7.08 (2 H, (R,S)-(E)-N-(3-hydroxybenzyl)-3-(4-hydroxy-3-

d, J 8.7), 7.03 (2 H, d, J 8.4), 7.03 (3 H, s), 6.77 (2 H, d, J 8.2), 6.59 methoxyphenyl)-N-(1-(4-hydroxyphenyl)-2-(methylamino)-2-

(2 H, d, J 8.5), 6.52 (2 H, d, J 8.6), 6.36 (2 H, d, J 8.8), 6.23 (1 H, s, oxoethyl)acrylamide 1q. δ_H(300 MHz, DMSO-d6, 90 °C) (Note:

CH), 2.12 (6 H, s, CH₃Ar), 1.35 (9 H, s, C(CH₃)₃). HPLC (see the 4 phenolic OH exchange with H₂O contained in the solvent

supplementary information) showed a purity of 100%.

(R,S)-(E)-N-(3-Hydroxybenzyl)-N-(1-(4-hydroxyphenyl)-2-((4-

hydroxyphenyl)amino)-2-oxoethyl)-3-(4-hydroxyoxy-3-

giving a very broad signal at around 9 ppm): 7.71 (1 H, s, NH),

7.39 (1 H, d, J 15.3, ArCH=CH), 7.11 (2 H, d, J 8.1), 7.00-6.87 (3

H, m), 6.75 (1 H, d, J 8.1), 6.68 (2 H, d, J 8.7), 6.67-6.46 (4 H, m),

methoxyphenyl)acrylamide 1l. δ_H(300 MHz, DMSO-d6, 90 °C) 6.02 (1 H, s, CH), 4.76, 4.53 (2 H, AB syst., J 17.1, CH₂Ar), 3.77 (3

(Note: the 4 phenolic OH exchange with H₂O contained in the H, s, OCH₃), 2.63 (3 H, d, J 4.5, CH₃N). HPLC (see supplementary

solvent giving a very broad signal at around 9 ppm): 9.63 (1 H, information) showed a purity of 99%.

s, NH), 7.41 (1 H, d, J 15.3, ArCH=CH), 7.33 (2 H, d, J 8.7), 7.17 (2

Biophysical and Biochemical tests

H, d, J 8.5), 7.00-6.85 (3 H, m), 6.75 (2 H, d, J 8.1), 6.73-6.63 (4

H, m), 6.58 (1 H, s), 6.55-6.48 (2 H, m), 6.17 (1 H, s, CH), 4.79 and

4.53 (2 H, AB syst., J 17.1, CH₂Ar), 3.76 (3 H, s, OCH₃). HPLC (see

supplementary information) showed a purity of 92%.

UV Spectroscopy

Synthetic polyphenols stock solutions were obtained by

dissolving the compounds in 100% dimethyl sulfoxide (DMSO;

Sigma) at given concentration (1.25 – 50 mM). Work solutions

were prepared diluting the appropriate stock solution in PBS

(150 mM, pH 7.4) at 12.5 – 500 µM, in such a manner that each

tube contained 1% of stock solution in DMSO. Solubility and

turbidity of polyphenols in function of the concentration was

determined by spectrophotometric measures using Shimadzu

UV-2700 Spectrophotometer and reading the absorbance

respectively at characteristic wavelength of each polyphenols

and at λ = 405 nm.

(R,S)-(E)-N-(2-(4-hydroxyphenyl)ethyl)-N-(1-(4-

hydroxyphenyl)-2-(methylamino)-2-oxoethyl)-3-(4-hydroxy-3-

methoxyphenyl)acrylamide 1m. δ_H(300 MHz, DMSO-d6, 90 °C)

(Note: the 4 phenolic OH exchange with H₂O contained in the

solvent giving a very broad signal at around 6 ppm): 7.73 (1 H,

broad s, NH), 7.44 (1 H, d, J 15.3, ArCH=CH), 7.21-7.14 (3 H, m),

7.05 (1 H, dd, J 8.2, 1.9), 6.87-6.74 (6 H, m), 6.63 (2 H, d, J 8.4),

6.01 (1 H, s, CH), 3.85 (3 H, s, OCH₃), 3.64-3.40 (2 H, m, CH₂N),

2.66 (3 H, d, J 4.5, CH₃NH), 2.63-2.48 (1 H, m, ArCHH), 2.18-2.03

(1 H, m, ArCH₂). HPLC (see supplementary information) showed

a purity of 92%.

Aβ sample preparation

One milliliter of DMSO was added to 1 mg of lyophilized

synthetic peptide (Aβ1-42, AβpE3-42 AnaSpec), reaching a final

concentration of 1 mg/mL. Aliquots of 75 μL were lyophilized

and stored at −20°C until used. For all experiments, stock

peptides were reconstituted as reported.⁵⁴The concentration

of the peptide in the stock solution was estimated using a molar

extinction coefficient at 214 nm, by Shimadzu UV-2700

Spectrophotometer.⁵⁵

(R,S)-(E)-N-benzyl-3-(4-hydroxy-3-methoxyphenyl)-N-(2-(tert-

butylamino)-1-(4-hydroxy-3-methoxyphenyl)-2-

oxoethyl)acrylamide 1n. After the treatment with the resin and

evaporation, in this case the residue was further purified by

chromatography (PE / AcOEt 6:4). δ_H(300 MHz, DMSO-d6, 90

°C): 9.00 (1 H, s, OH), 8.57 (1 H, s, OH), 7.45 (1 H, broad s, NH),

7.42 (1 H, d, J 15.3, ArCH=CH), 7.21-6.99 (6 H, m), 6.94 (1 H, d, J

8.0), 6.84-6.66 (4 H, m), 6.70 (1 H, d, J 15.3, ArCH=CH), 5.97 (1

18 | J. Name., 2012, 00, 1-3

Please do not adjust margins

Page 19 of 20

Organic & Biomolecular Chemistry

Please do not adjust margins

Organic and Biomolecular Chemistry

ARTICLE

For the preparation of the working samples, stock solution of 6.

each peptide was divided in two or more aliquots. One was

diluted to 5 μM in PBS containing 1% (v/v) DMSO to have a

reference sample, the others were diluted in PBS containing the 7.

appropriate quantity of polyphenols stock solution in DMSO in

such a manner that each samples contains 1% of DMSO. Final 8.

pH was measured and eventually corrected at 7.4 with few μL

of 1M HCl.

M. Hirohata, K. Hasegawa, S. Tsutsumi-Yasuhara, Y.

Ohhashi, T. Ookoshi, K. Ono, M.^DY^Oa^I:m¹⁰a^.d¹⁰a³⁹a^/n^Cd^7OH^B.⁰N²¹a⁸i²k^Hi,

Biochemistry, 2007, 46, 1888-1899.

V. L. N. Ngoungoure, J. Schluesener, P. F. Moundipa and H.

Schluesener, Mol. Nutr. Food Res., 2015, 59, 8-20.

N. Ferreira, I. Cardoso, M. R. Domingues, R. Vitorino, M.

Bastos, G. Bai, M. J. Saraiva and M. R. Almeida, FEBS

Lett., 2009, 583, 3569-3576.

View Article Online

Thioflavine T Fluorescence Spectroscopy

9.

M. Yamada, K. Ono, T. Hamaguchi and M. Noguchi-

Shinohara, in Natural Compounds as Therapeutic Agents

for Amyloidogenic Diseases, ed. N. Vassallo, Springer

International Publishing, Cham, 2015, pp. 79-94.

K. M. Pate, M. Rogers, J. W. Reed, N. van der Munnik, S. Z.

Aβ peptides (5 μM) were incubated at 37 °C in presence

/absence of polyphenols as previous described and analyzed in

parallel. ThT fluorescence was followed in time during

aggregation. For this purpose, 47.5 μl of Aβ with and without 10.

test compounds, were mixed with 2.5 μL ThT (400 μM) in a 3

mm path length fluorescence cuvette. ThT fluorescence was

measured by Luminescence Spectrometer Perkin Elmer LS50B 11.

at excitation and emission wavelengths of 440 nm (slit width=5

nm) and 482 nm (slit width = 10 nm), respectively.

Vance and M. A. Moss, Cns Neurosci. Therap., 2017, 23

,

135-144.

R. Randino, M. Grimaldi, M. Persico, A. De Santis, E. Cini,

W. Cabri, A. Riva, G. D'Errico, C. Fattorusso, A. M. D'Ursi

and M. Rodriquez, Sci Rep, 2016, 6.

Transmission electron microscopy (TEM)

Aβ peptides (10 µM) were separately co-incubated with single

12.

T. T. An, S. Feng and C. M. Zeng, Redox Biol., 2017, 11, 315-

321.

polyphenol at molar ratio 1:5 (Aβ:polyphenol) in sterile 13.

microtubes. To evaluate the morphology and the sizes of the

species in the different samples, 5 μL of each one were

adsorbed for 5 min onto carbon coated 300-mesh copper grids.

The aggregates species were negatively stained for 1 min with 5

μL of 1% Uranyl Acetate. All air-dried specimens were examined

with a Zeiss LEO 900 electron microscope (Zeiss, Stuttgart, 14.

Germany) operating at 80 kV. Images flattening and analysis

J. M. Ringman, S. A. Frautschy, E. Teng, A. N. Begum, J.

Bardens, M. Beigi, K. H. Gylys, V. Badmaev, D. D. Heath,

L. G. Apostolova, V. Porter, Z. Vanek, G. A. Marshall, G.

Hellemann, C. Sugar, D. L. Masterman, T. J. Montine, J.

L. Cummings and G. M. Cole, Alzheimer's Res. Ther.,

2012, 4, 43.

S. Feng, X.-H. Song and C.-M. Zeng, FEBS Lett., 2012, 586

,

3951-3955.

was performed by ImageJ software.

15.

S. Montanari, M. Bartolini, P. Neviani, F. Belluti, S. Gobbi,

L. Pruccoli, A. Tarozzi, F. Falchi, V. Andrisano, P. Miszta,

A. Cavalli, S. Filipek, A. Bisi and A. Rampa,

ChemMedChem, 2016, 11, 1296-1308.

Conflicts of interest

16.

L. A. Wessjohann, G. Kaluderovic, R. A. W. Neves Filho, M.

C. Morejon, G. Lemanski and T. Ziegler, in Science of

Synthesis: Multicomponent Reactions, Vol. 1, ed. T. J. J.

Müller, 2013, pp. 415-497.

L. Banfi, R. Riva and A. Basso, Synlett, 2010, 23-41.

M. Spallarossa, L. Banfi, A. Basso, L. Moni and R. Riva, Adv.

Synth. Cat., 2016, 358, 2940-2948.

L. Moni, C. F. Gers-Panther, M. Anselmo, T. J. J. Muller and

R. Riva, Chem. Eur. J., 2016, 22, 2020-2031.

S. Caputo, A. Basso, L. Moni, R. Riva, V. Rocca and L. Banfi,

Beilstein J. Org. Chem., 2016, 12, 139-143.

There are no conflicts to declare

Acknowledgements

17.

18.

This study is supported by Fondazione Cariplo, under

"Integrated Biotechnology and Bioeconomy" programme.

We thank Valeria Rocca for HPLC analyses, Giuliana Ottonello

for HRMS, Giulia Baruzzo, Gianluca Pusceddu, and Riccardo

Loddo for their experimental contribution to this work.

19.

20.

21.

22.

23.

E. Tassano, A. Alama, A. Basso, G. Dondo, A. Galatini, R.

Riva and L. Banfi, Eur. J. Org. Chem., 2015, 6710-6726.

D. Goyal, S. Shuaib, S. Mann and B. Goyal, Acs Combi. Sci.,

2017, 19, 55-80.

References

1.

2.

3.

4.

A. J. Doig and P. Derreumaux, Curr. Opin. Struct. Biol.,

2015, 30, 50-56.

Q. Nie, X. G. Du and M. Y. Geng, Acta Pharm. Sinica, 2011,

32, 545-551.

F. Belluti, A. Rampa, S. Gobbi and A. Bisi, Expert Opin. Ther.

Pat., 2013, 23, 581-596.

C. Ran, X. Xu, S. B. Raymond, B. J. Ferrara, K. Neal, B. J.

Bacskai, Z. Medarova and A. Moore, J. Amer. Chem. Soc.,

2009, 131, 15257-15261.

E. Elhalem, B. N. Bailey, R. Docampo, I. Ujváry, S. H.

Szajnman and J. B. Rodriguez, J. Med. Chem., 2002, 45

,

3984-3999.

24.

25.

S. W. Youn and E. M. Lee, Org. Lett., 2016, 18, 5728-5731.

B. Schmidt, N. Elizarov, R. Berger and F. Holter, Org.

Biomol. Chem., 2013, 11, 3674-3691.

C. D'Arrigo, M. Tabaton and A. Perico, Biopolymers, 2009,

91, 861-873.

26.

27.

5.

Y. Porat, A. Abramowitz and E. Gazit, Chem. Biol. Drug

Des., 2006, 67, 27-37.

C. Dammers, M. Schwarten, A. K. Buell and D. Willbold,

Chem. Sci., 2017, 8, 4996-5004.

J. Name., 2013, 00, 1-3 | 19

Please do not adjust margins

Organic & Biomolecular Chemistry

Page 20 of 20

ARTICLE

Organic and Biomolecular Chemistry

28.

29.

S. Schilling, T. Hoffmann, S. Manhart, M. Hoffmann and H. 54.

U. Demuth, FEBS Lett., 2004, 563, 191-196.

S. Schilling, T. Lauber, M. Schaupp, S. Manhart, E. Scheel,

D. Galante, A. Corsaro, T. Florio, S. Vella, A. Pagano, F.

Sbrana, M. Vassalli, A. Perico and C. D'Arrigo, Int. J.

Biochem. Cell Biol., 2012, 44, 2085-2093.

View Article Online

DOI: 10.1039/C7OB02182H

G. Bohm and H. U. Demuth, Biochemistry, 2006, 45

12393-12399.

D. Galante, F. S. Ruggeri, G. Dietler, F. Pellistri, E. Gatta, A.

Corsaro, T. Florio, A. Perico and C. D'Arrigo, Int. J.

Biochem. Cell Biol., 2016, 79, 261-270.

,

55.

L. W. Hung, G. D. Ciccotosto, E. Giannakis, D. J. Tew, K.

Perez, C. L. Masters, R. Cappai, J. D. Wade and K. J.

Barnham, J. Neurosci., 2008, 28, 11950-11958.

30.

31.

32.

33.

34.

Y. Liu, S. H. Wang, S. Z. Dong, P. Chang and Z. F. Jiang, Rsc

Adv., 2015, 5, 62402-62413.

F. Ding, J. M. Borreguero, S. V. Buldyrey, H. E. Stanley and

N. V. Dokholyan, Proteins, 2003, 53, 220-228.

A. Sgarbossa, D. Giacomazza and M. di Carlo, Nutrients,

2015,

S. Sinha, D. H. J. Lopes and G. Bitan, Acs Chem. Neurosci.,

2012, , 473-481.

7, 5764-5782.

3

35.

36.

E. Gazit, Faseb J., 2002, 16, 77-83.

C. Soto, E. M. Sigurdsson, L. Morelli, R. A. Kumar, E. M.

Castano and B. Frangione, Nat. Med., 1998, 4, 822-826.

37.

38.

M. Convertino, R. Pellarin, M. Catto, A. Carotti and A.

Caflisch, Protein Sci., 2009, 18, 792-800.

M. Benchekroun, A. Romero, J. Egea, R. Leon, P.

Michalska, I. Buendia, M. L. Jimeno, D. Jun, J. Janockova,

V. Sepsova, O. Soukup, O. M. Bautista-Aguilera, B.

Refouvelet, O. Ouari, J. Marco-Contelles and L. Ismaili, J.

Med. Chem., 2016, 59, 9967-9973.

39.

40.

H. Josien, S. B. Ko, D. Bom and D. P. Curran, Chem. Eur. J.,

1998, 4, 67-83.

M. Suzuki, J. F. K. Kotyk, S. I. Khan and Y. Rubin, J. Am.

Chem. Soc., 2016, 138, 5939-5956.

41.

42.

43.

W. Gu and R. B. Silverman, Org. Lett., 2003, 5, 415-418.

S. Imai and H. Togo, Tetrahedron, 2016, 72, 6948-6954.

A. J. Harnoy, G. Slor, E. Tirosh and R. J. Amir, Org. Biomol.

Chem., 2016, 14, 5813-5819.

44.

45.

46.

J. Hoffmann and U. Kazmaier, Angew. Chem., Int. Ed.,

2014, 53, 11356-11360.

R. Jaiswal, M. H. Dickman and N. Kuhnert, Org. Biomol.

Chem., 2012, 10, 5266-5277.

L. Jiménez-González, S. García-Muñoz, M. Álvarez-Corral,

M. Muñoz-Dorado and I. Rodríguez-García, Chem. Eur.

J., 2007, 13, 557-568.

47.

48.

49.

J. Liu, K. Wu, T. Shen, Y. Liang, M. Zou, Y. Zhu, X. Li, X. Li

and N. Jiao, Chem. Eur. J., 2017, 23, 563-567.

M. Kim, W. B. Euler and W. Rosen, J. Org. Chem., 1997, 62

,

3766-3769.

A. Massink, J. Louvel, I. Adlere, C. van Veen, B. J. H.

Huisman, G. S. Dijksteel, D. Guo, E. B. Lenselink, B. J.

Buckley, H. Matthews, M. Ranson, M. Kelso and A. P.

Ijzerman, J. Med. Chem., 2016, 59, 4769-4777.

T. Mäkelä, J. Matikainen, K. Wähälä and T. Hase,

Tetrahedron, 2000, 56, 1873-1882.

C. D. Selassie, C. Hansch, T. A. Khwaja, C. B. Dias and S.

Pentecost, J. Med. Chem., 1984, 27, 347-357.

G. H. Lee, E. B. Choi, E. Lee and C. S. Pak, J. Org. Chem.,

1994, 59, 1428-1443.

50.

51.

52.

53.

T. Ventrice, E. M. Campi, W. R. Jackson and A. F. Patti,

Tetrahedron, 2001, 57, 7557-7574.

20 | J. Name., 2012, 00, 1-3

Please do not adjust margins

Article Doi

Multicomponent, fragment-based synthesis of polyphenol-containing peptidomimetics and their inhibiting activity on beta-amyloid oligomerization

DOI: 10.1039/c7ob02182h

Source and publish data:

Authors:

Article abstract of DOI:10.1039/c7ob02182h

Full text of DOI:10.1039/c7ob02182h

Products guided by the article

R&D Labs maybe for 854261-52-2

Relevant to this article

Hot Product