Bioinspired design for the assembly of Glypromate neuropeptide conjugates with active pharmaceutical ingredients

NJC

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Bioinspired design for the assembly of

s

Glypromate neuropeptide conjugates

Cite this: New J. Chem., 2020,

with active pharmaceutical ingredients†

44, 21049

a

b

c

Sara C. Silva-Reis,

A. Catarina V. D. dos Santos,

Xerardo Garcıa-Mera,

´

a

Jose E. Rodrıguez-Borges

and Ivo E. Sampaio-Dias

*

´

Neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases, are a class of heterogeneous

pathologies of the central nervous system (CNS) aﬀecting millions of people worldwide. CNS-related

pathologies represent a global health burden in developed and developing countries with no curative

treatments currently available. Thus, the development of novel multitarget neuroprotective drugs is a

health priority. In this work, a bioinspired methodology in solution-phase for the assembly and

s

regioselective conjugation of Glypromate neuropeptide with active pharmaceutical ingredients (APIs) is

described. The main purpose is to design new hybrid molecules which may oﬀer increased systemic

s

resistance of Glypromate towards proteases and/or allow the controlled release of both APIs and

the neuroprotective peptide within CNS. For the synthesis of such peptide-hybrid compounds

s

(R)-1-aminoindane, amantadine, and memantine were selected as APIs for conjugation with Glypromate .

s

Furthermore, capping strategies are explored to prepare Glypromate conjugates with more favorable

pharmacodynamic profiles by masking polar exposed groups. Overall, this synthetic approach led to the

development of a small library of 12 conjugates with improved drug-like properties in comparison with

s

Received 2nd October 2020,

Accepted 20th November 2020

Glypromate , paving the way for the discovery of novel CNS multitarget drugs. Additionally, by exploring

the bis-functionalization of glutamate, the formation of chiral glutarimides is disclosed for the first time

employing TBTU as the coupling reagent. This unusual reactivity of TBTU with glutamate oﬀers a new

synthetic approach for the preparation of chiral glutarimide alkaloids.

DOI: 10.1039/d0nj04851h

rsc.li/njc

previously thoroughly studied and demonstrated to be clini-

cally safe in humans is considered a privileged methodology

Introduction

Neurodegenerative diseases (NDD) are characterized by the slow for the eﬀective repositioning of known drugs through the

and gradual degeneration of Central Nervous System (CNS) development of novel potential bioactive compounds with

4

neurons, ultimately resulting in neuronal death. Currently, the improved biological and pharmacological profiles. Among

1

existing therapeutic approaches only address the symptoms.

these strategies, chemical conjugation of bioactive scaﬀolds is

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are commonly employed in the development of CNS drugs to

the top two common NDD with an estimated 50 million people achieve synergistic or complementary eﬀects. A paradigmatic

worldwide living with AD or another type of dementia and example is Ladostigil, which combines the neuroprotective

2

6

.2 million with PD. Predictions are worrisome, pointing to 82 eﬀects of the selective monoamine oxidase (MAO)-B inhibitor

2

s

and 8.7 million living with dementia and PD by 2030, respectively. rasagiline (Azilect ) with the cholinesterase inhibitory (AChE)

5

The scarcity of multifactorial therapeutic approaches to activity of rivastigmine. Further studies with the conjugation

manage CNS-related pathologies makes the development of of two or more anti-Alzheimer’s drugs (tacrine, donepezil and

3

novel drugs an imperative. The rescuing of drugs that were rivastigmine) show promising results. For example, tacrine,

6

which is a potent AChE inhibitor (IC50 = 167 nM), was the first

a

7

LAQV/REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences,

University of Porto, 4169-007 Porto, Portugal. E-mail: idias@fc.up.pt

Institute of Chemical Technologies and Analytics, TU Wien, 1060 Vienna, Austria

Department of Organic Chemistry, Faculty of Pharmacy, University of Santiago de

Compostela, E-15782 Santiago de Compostela, Spain

FDA-approved anti-AD drug in 1993, but was soon withdrawn

8

from the market due to hepatotoxicity issues. This prompted the

b

c

development of multifunctional anti-AD drugs, such as tacrine

conjugates with a 1-azabenzanthrone moiety, which showed

higher AChE inhibitory activity than the parent drug (within the

1

13

1

†

Electronic supplementary information (ESI) available: NMR spectra ( H, C{ H}

7,9

and DEPT-135) for all the synthesized compounds. See DOI: 10.1039/d0nj04851h

nanomolar range) and also anti-Ab aggregation activity.

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s

According to literature, the metabolism of Glypromate

22

occurs from C- to N-terminal, promoted by carboxipeptidases.

In this sense, it is reasonable to consider the C-terminal amino

acid residue, glutamic acid, as the most adequate site for deriva-

s

tization and assembly of Glypromate conjugates with active

pharmaceutical ingredients (APIs). This strategy may allow the

development of conjugates with improved resistance towards

enzymatic degradation and/or the controlled release of APIs

s

.

Fig. 1 Molecular structure of Glypromate

s

and Glypromate , oﬀering the possibility to tune the balance

of hydrophobicity/hydrophilicity to selectively promote the

Insulin-like growth factor-I (IGF-I), also known as somato- delivery across the BBB. It is known that the functionalization

medin C, is a 70-mer peptide hormone that acts as an essential at the C-terminal of this peptide, for example by converting

factor for the development and maturation of the CNS and the g-carboxylic acid into a carboxamide or by reducing it into

which is also important to neuroplasticity. However, its use as the corresponding primary alcohol prevented neuronal cell

s

therapeutic agent is restricted due to its poor ability to cross the death at 1 mM, the same concentration at which Glypromate

1

0,11

18,19

blood–brain barrier (BBB).

This protein is cleaved by typically display neuroprotective eﬀects.

Moreover, con-

protease-mediated metabolism into des-N-(1-3)-IGF-I and version of both a and g-carboxylic acid moieties into methyl

the N-terminal tripeptide glycyl-L-prolyl-L-glutamic acid (also ester functional groups has proved to improve lipophilicity

s

known as GPE or Glypromate , Fig. 1).

without compromising the neuroprotective activity of the

s

18

Glypromate is a promising lead compound candidate for peptide at 1 mM. Taken together, this data indicates that

clinical therapies and the metabolite of this tripeptide, Glypromate is quite tolerant to functionalization at the

s

cyclo(Pro-Gly), (obtained by the cleavage from C- to N-terminal) glutamate residue.

12

s

is also a small, stable and bioactive molecule in the CNS.

The selection of APIs for conjugation with Glypromate is of

s

Glypromate stimulates acetylcholine and dopamine release in utmost importance in order to balance its high hydrophilicity,

rat cortex and has neuroprotective properties from hypoxia- which is the main cause for the low oral absorption associated

13–15

23,24

ischemia and glutamate induced injuries.

with this peptide.

In this sense, APIs should be hydro-

s

The therapeutic potential of this short peptide for CNS- phobic enough to tower over the hydrophilicity of Glypromate

s

related pathologies led Glypromate to clinical trials by Neuren and not exceed 180 Da, in order to respect the Lipinski’s rule of

2

5

Pharmaceuticals to study its eﬀects on brain impairment after five concerning the molecular mass of the conjugates. Hence,

1

6

s

major heart surgery. Data suggest that Glypromate inhibits accordingly to lipophilicity and molecular mass parameters,

caspase III dependent apoptosis and despite its excellent safety three APIs used in clinic for neurodegenerative conditions were

profile, in December 2008, the company discontinued selected for conjugation with this neuropeptide: (R)-1-amino-

s

Glypromate research after the completion of phase III of indane, amantadine, and memantine. Additionally, to further

clinic trials due to lack of evidence of brain improvement after increase the hydrophobicity of the conjugates, exposed polar

1

7

major heart surgery.

Nonetheless, the neuroprotective properties of Glypromate

groups should be masked. To meet this goal, both N-terminal

amino group and C-terminal carboxylic acid should be converted

s

are being explored by the development of structurally-related into tertiary amine and ester moieties, respectively. Considering

1

2,13,18–20

26

analogues

and hybrid molecules containing that N,N-dimethylglycine is known to decrease oxidative stress,

s 21

Glypromate . Cacciatore and co-workers reported the first conversion of N-terminal glycine residue into its N,N-dimethylated

successful Glypromate conjugate with lipoic acid (LA) to analogue may confer protection against oxidative stress while

generate a LA-Glypromate hybrid compound. LA-Glypromate

not only demonstrated neuroprotective properties against

H O and 6-hydroxydopamine (6-OHDA) but also high CNS Rasagiline (Azilect ), a pharmaceutical used in PD therapy as a

permeability. These results show that Glypromate can be MAO-B irreversible inhibitor. This metabolite decreases apop-

successfully covalently conjugated without loss of its intrinsic totic processes and displays a good neuroprotective profile.

neuroprotective activity. To the best of our knowledge, despite Amantadine (b, Fig. 2) is commercialized as Symmetrel and

s

21

s

improving the lipophilicity of the conjugates.

(R)-1-Aminoindane (a, Fig. 2) is the principal metabolite of

s

2

1

s

27

2

7

s

the success of the repurposing strategy for the rescue of CNS employed in PD therapy as a non-competitive antagonist

s

active drugs, only one successful conjugate of Glypromate has of NMDA receptors, leading to an increase in the amount of

2

1

28

been reported so far in literature.

endogenous dopamine. Memantine (c, Fig. 2) is the API of

s

The scarcity of such hybrid Glypromate molecules makes Namenda and is used in AD therapy as an antagonist of NMDA

imperious the establishment of a reliable methodology for the receptors.

preparation of valuable multitarget compounds for CNS-related In this work, the conjugates (I–VI) depicted in Fig. 2 were

29

pathologies. In this sense, we sought to contribute for designed in accordance with the goals laid out in this intro in

the bioinspired design and the development of an eﬃcient order to explore suitable synthetic routes for the unprecedented

s

methodology for the assembly of Glypromate conjugates in development peptide-hybrid molecules of Glypromate with

solution-phase.

different APIs by covalent conjugation at the C-terminal site.

2

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s

Fig. 2 Glypromate conjugates I–VI.

Results and discussion

both glutamate derivatives with APIs a-c was performed by

peptide coupling using standard protocols in solution-phase,

namely 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetra-

fluoroborate (TBTU) and triethylamine as the coupling reagent and

The synthesis of conjugates I, II, IV and V (Fig. 2) started with

the functionalization of the glutamate residue at either the a or

the g carboxylic acid positions, in order to aﬀord monofunctio-

nalized glutamates. For this purpose, the synthesis started with

the mono functionalization of commercially available methyl

ester glutamate derivatives (Scheme 1).

The use of mono methyl ester glutamates (Boc-Glu(OH)-OMe

and Boc-Glu(OMe)-OH, Scheme 1) serves two purposes: it

allows for the regioselective preparation of monofunctionalized

glutamates and at same time the methyl ester moiety acts as a

capping strategy for the carboxylic acid. Functionalization of

30

tertiary base, respectively. Functionalized glutamates 1(a-c) and

3(a-c) were obtained in excellent yields after chromatographic

purification (89–95%, Scheme 1). Subsequently, the removal of

tert-butyloxycarbonyl (Boc) group was performed by acidolysis

using trifluoroacetic acid (TFA), delivering glutamates 2(a-c)

and 4(a-c) as ammonium trifluoroacetates with near quantitative

yield (97–98%) without the need of chromatographic purification

(Scheme 1).

With monofunctionalized glutamate derivatives in hand,

bis-functionalization using Boc-Glu(OH)-OH was explored

(Scheme 2).

To accomplish this task, two equivalents of APIs and TBTU

were used instead. During the reactions, the formation of two

new spots on the TLC were detected, which we initially attributed

to the presence of mono and bis-functionalized products. After

the addition of an excess of the APIs the reaction mixture

remained unchanged, which led us to isolate both compounds

in order to identify the products formed. As suspected, the least

polar compounds (R E 0.9 in EtOAc) were identified (NMR) as

f

the bis-functionalized glutamates 5(a-c), albeit with unexpected

low yields (25–35%) in contrast with the yields reported for the

mono-functionalized counterparts. Besides glutamates 5(a-c),

f

other by-products (R E 0.6 in EtOAc) were isolated. NMR analysis

(Fig. S31–S36, ESI†) allowed the unequivocal assignment of

these by-products as N-substituted (S)-3-aminopiperidine-2,6-

diones, glutarimides 6(a-c). Chiral glutarimides 6(a-c) were

isolated in comparable yields (19–30%) to those reported for

bis-functionalized glutamates. To the best of our knowledge, the

preparation of chiral glutarimides from glutamate using TBTU is

unprecedented. A possible mechanism for the formation of

glutarimides 6(a-c) mediated by TBTU is proposed in Scheme 3.

Scheme 1 Synthesis of functionalized glutamates 2(a-c) and 4(a-c).

Conditions and reagents: (i) Et

ii) TFA, anhydrous CH Cl

3 2 2

N, TBTU, API (a-c), anhydrous CH Cl ;

(

2

.

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7(a-c) and 8(a-c), respectively, was performed using a robust

and highly eﬃcient one-pot protocol in solution-phase devel-

3

1,32

oped in our research group.

Briefly, the methodology con-

sists in three essential steps (Scheme 4): (A) peptide coupling of

proline (via a tertiary base like Et N) with pre-activated glycine

3

(using O-succinimidyl ester derivative, Boc-Gly-OSu); (B) in situ

activation of the amide-carboxylate intermediate to the corres-

ponding active ester via TBTU and (C) coupling of the final

amino acid by aminolysis between the functionalized gluta-

mates, 2(a-c) or 4(a-c), and the active esters generated in step B.

This one-pot protocol has several advantages such as: (1) no

need for isolation of the intermediates; (2) reduction of

chemical waste (highly competitive in terms of global E-factor,

being superior to solid-phase peptide synthesis (SPPS), classical

solution-phase synthesis and mechanochemical approaches such

32

Scheme 2 Synthesis of bis-functionalized glutamates 5(a-c) and chiral as ball-milling); (4) faster assembly of peptides in comparison

glutarimides 6(a-c). Conditions and reagents: (i) Et N, TBTU, API (a-c),

anhydrous CH Cl

32

3

with classical iterative approaches; and (5) high overall yield.

2

.

Additionally, in contrast with SPPS, this methodology allows for

the use of bis-functionalized glutamate, which is not attainable by

Despite the feasibility of the synthesis of bis-functionalized SPPS since one of the carboxylic acids is covalently linked to the

20

glutamates 5(a-c), along with the formation of chiral glutar- resin.

imides 6(a-c), in this work, these glutamates were not further

explored for the assembly of Glypromate conjugates III/VI due were readily prepared in good overall yields (51–70%), as shown

to violation of the Lipinski’s rule of 5, namely molecular weight in Scheme 4. Conjugates 8(a-c) were obtained in lower yields

Following this protocol, N-Boc conjugates 7(a-c) and 8(a-c)

s

over 500 Da (Table S1 of ESI†).

(51–69%) in comparison with 7(a-c), which is explained by a

Starting from glutamates 2(a-c) and 4(a-c), the assembly higher steric hindrance of the glutamates 4(a-c) in contrast with

s

of the corresponding Glypromate conjugates intermediates, 2(a-c). Note that APIs are covalently linked at a-carboxylate in

Scheme 3 Mechanism proposed for the synthesis of chiral glutarimides 6(a-c) mediated by TBTU.

2

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Scheme 4 One-pot assembly of conjugates 7(a-c) and 8(a-c).

Scheme 6 Iterative route for the preparation of conjugate Vb. Conditions

and reagents: (i) Et

3

N, TBTU, anhydrous CH

2

Cl

2

; (ii) TFA, anhydrous

Cl

Scheme 5 Preparation of conjugates I(a-c) and II(a-c). Conditions and

reagents: (i) TFA, anhydrous CH Cl .

2 2

CH Cl ; (iii) Et N, TBTU, N,N-dimethylglycine, anhydrous CH

2

3

2

.

3

CHCl , EtOAc; stoichiometry: 1–2 equiv. of each reagent)

4(a-c), while in 2(a-c) they are attached at g-carboxylate. Steric

to synthesize conjugate Vb by coupling dipeptide 10b with

N,N-dimethylglycine were unsuccessful (Scheme 6).

hindrance in 4(a-c) is therefore responsible for slowing down

the coupling rates, and hence the yields.

To circumvent this hurdle, reductive amination was

attempted by reacting conjugates I/II with formaldehyde using

The final compounds I(a-c) and II(a-c) were successfully

obtained in practically quantitative yields after cleavage of the

N-protecting group by acidolysis using TFA (Scheme 5).

The diﬀerence between conjugates I/II and IV/V lies on the

presence of a tertiary amine (N,N-dimethylglycyl residue) in the

latter ones. Since the N,N-dimethylglycine O-succinimidyl ester

is not commercially available, the preparation of type IV and V

conjugates following the classical iterative synthesis approach

in solution-phase was envisioned. This synthetic route was

tested for the preparation of conjugate Vb (Scheme 6). For this

purpose, Boc-Pro-OH was coupled with glutamate 4b using the

NaBH(OAc) as the reducing agent (Scheme 7).

3

After the reductive amination and subsequent removal of

the solvent by distillation, the excess of the reductive agent was

precipitated upon addition of CH Cl . Following this workup,

2 2

conjugates IV and V were isolated in good yields (60–75%)

without the need of chromatographic purifications, except for IVa.

Druglike properties and predicted BBB permeability of the

s

Glypromate conjugates

chemistry of TBTU, aﬀording the corresponding dipeptide 9b Evaluation of the physicochemical properties for the novel drug

in excellent yield (91%). Successful removal of the carbamate candidates is of great importance to estimate qualitatively oral

was performed by acidolysis using TFA, delivering the ammo- bioavailability and membrane permeability. Considering the

nium trifluoroacetate 10b in practically quantitative yield parameters originally proposed by Lipinski and co-workers

(98%). All the attempts (temperature: 0–40 1C, solvents: CH

2

Cl

2

,

[molecular weight (MW), partition coeﬃcient (clog P), number

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lipophilicity (conjugates type IV and V, respectively). Apart from

conjugates with (R)-1-aminoindane (IVa and Va), all the remaining

N,N-dimethylated conjugates display high clog P values (clog P 4 1)

+

t

within the range of adequate CNS drug’s clog P (o5). PSA is a

medicinal chemistry metric for evaluation and optimization of a

molecule’s ability to permeate cells. Molecules with high PSA

higher than 140 A ) tend to display low ability to permeate cell

membranes. For molecules intended to cross the BBB, a PSA less

36

t

2

(

t

2

35

2 35,36

than 90 A is usually required, ideally in the range of 60–70 A .

In silico BBB permeation shows that all the conjugates are

considered as BBB+ (BB ratio Z0.1). Except for conjugates Ia,

Ic, IIa and IIc, all the remaining conjugates display enhanced

BBB permeation (BB ratio ranging between 0.535–0.739) com-

paratively to the parent compound (BB ratio of 0.517). Among

the series of compounds, conjugates IVb and Vb are considered

t

the most promising (high clog P: 1.61 and 1.30, low PSA: both

with 108.05 A , high BB ratio: 0.655 and 0.739, respectively).

Scheme 7 Preparation of conjugates IV(a-c) and V(a-c) by reductive

amination of I(a-c) and II(a-c), respectively. Conditions and reagents:

2

3

i. Formaldehyde solution 37 wt% in water, NaBH(OAc) , 1,2-dichloroethane.

Characterization section

of H-bond acceptors (HBA), number of H-bond donors

3

s

(

HBD)], together with the parameters later suggested by Veber During the characterization of the Glypromate conjugates it

t

2

and co-workers [topological polar surface area ( PSA in Å ), was observed that some of these compounds display a splitting

1

13

number of rotatable bonds (nrotb), sum of H-bond donors and pattern in both H and C-NMR spectra at room temperature.

3

4

acceptors]

conjugates I(a-c), II(a-c), IV(a-c), V(a-c) and Glypromate . The (rotamers). It is known that prolyl-containing peptides such

the druglike properties were calculated for This phenomenon is attributed to cis–trans isomerization

s

results obtained are listed in Table 1.

as Glypromate and its derivatives commonly exhibit rotamers

3

1,32

A close inspection of the clog P values obtained for the in solution.

The presence of rotamers can be easily proven

3

1,32

conjugates with free N-terminal, namely conjugates I(a-c) and using variable-temperature NMR (VT-NMR) experiments.

II(a-c), shows low lipophilicity (clog P o 1); the presence of Using these simple, albeit effective VT-NMR experiments, it is

(

R)-1-aminoindane in compounds Ia and IIa considerably possible to observe the coalescence of the signals as the

3

1,32

enhances the lipophilicity, albeit with negative clog P values temperature rises,

thus excluding epimerization events.

(

ꢀ0.69 and ꢀ1.00, respectively) in contrast with the parent Although the methodology employed for the assembly of con-

31

peptide (clog P = ꢀ4.39).

jugates is known to preserve the stereochemistry of peptides,

s

32

As expected, N,N-dimethylation of the glycine residue in and in particular for Glypromate and its analogues, VT-NMR

compounds type I and II leads to a substantial increment in experiments were performed for conjugate Ic as a representa-

tive example (Fig. 3).

1

In Fig. 3, two H-NMR spectra (DMSO-d , truncated for

6

clarity) of conjugate Ic are shown: one recorded at 26 1C (lower)

Table 1 Calculated druglike properties for conjugates I(a-c), II(a-c),

IV(a-c), V(a-c) and Glypromate

1

s

and the other at 100 1C (upper). In the H-NMR spectrum

recorded at 26 1C is observed the duplication of several signals

with 60 : 40 ratio. When the temperature is raised at 100 1C, those

signals undergo complete coalescence. This data unambiguously

a

rotb

t

a

2

b

Conjugate

MW

clog P HBA HBD

n

PSA /Å BB ratio

Ia

Ib

Ic

430.50 ꢀ0.69

448.56 0.39

476.62 0.51

430.50 ꢀ1.00

448.56 0.08

476.62 0.20

458.56 0.53

476.62 1.61

504.67 1.73

458.56 0.22

476.62 1.30

504.67 1.43

9

o7

4

2

5

o3

9

10

7

130.83 0.443

130.83 0.639

130.83 0.452

130.83 0.535

130.83 0.719

130.83 0.543

108.05 0.454

108.05 0.655

108.05 0.470

108.05 0.552

108.05 0.739

108.05 0.565

150.03 0.517

o90

IIa

IIb

IIc

IVa

IVb

IVc

Va

Vb

Vc

s

Glypromate 301.30 ꢀ4.39

+

35

CNS drugs o500 o5

o8

a

Properties calculated using Cheminformatics software [http://www.

b

molinspiration.com]. In silico BBB permeability using Cheminformatics

1

software [http://admet.scbdd.com/calcpre/index/], Category 0: BBBꢀ; Fig. 3 Sacked H-NMR spectra (truncated for clarity) of conjugate Ic

Category 1: BBB+; BB ratio Z0.1: BBB+; BB ratio o0.1: BBBꢀ.

6

(400 MHz, DMSO-d ) at 26 1C and 100 1C.

2

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shows that the origin of the splitting pattern found in the NMR at 400.15 MHz and 100.62 MHz, respectively. The H-NMR

spectra at 26 1C results from cis–trans rotamers.

spectra were calibrated using residual protic signals from

deuterated solvents (CDCl : d = 7.26, d_C= 77.16; CD OD:

3

H

3

7

d_H= 3.31, d_C= 49.00; DMSO-d : d = 2.50, d_C= 39.52) and

6

H

Conclusion

are reported in ppm. The nomenclature used for the assign-

ment of protons and/or carbons for each a-amino acid residue

in the peptide chains was made using a single letter system in

subscript for the amino acid residue (G: glycine; P: L-proline;

E: L-glutamic acid) and indicating the proton (or group of

protons) and/or the carbons in the structures by starting the

numeration at the carbonyl carbon of each a-amino acid

residue. Assignment of protons and/or the carbons relative to

the APIs scaﬀolds are indicated in subscript as follows:

Herein, the feasibility of a bioinspired methodology for the

assembly of Glypromate conjugates was proven with the

s

successful preparation of 12 novel conjugates with (R)-1-amino-

indane, amantadine and memantine. The key steps are (1) the

one-pot synthesis for the assembly of conjugates 7(a-c)/8(a-c)

and (2) the reductive amination for the preparation of conju-

gates IV/V from I/II, respectively.

s

Conjugation of Glypromate with neuroactive APIs and

capping of the exposed polar groups proved to be advantageous

(R)-1-aminoindane (a); amantadine (b); memantine (c). Optical

t

rotations were measured on a JASCO P-2000 thermostated

polarimeter using a sodium lamp and are reported as follows:

in regards with lipophilicity (clog P up to 1.73) and PSA

2

(

108.05–130.83 Å ) parameters, which compare favourably with

y

ꢀ1

t

2

D

a expressed in (1) (dm ) (g ), in which y is temperature in

the parent peptide (clog P = ꢀ4.39 and PSA = 150.03 Å ). In this

sense, conjugates I/II and IV/V are expected to display better

permeation across the BBB than Glypromate . Globally, among

the tested APIs, amantadine and memantine are considered the

most suitable for conjugation with Glypromate in terms of

Celsius and c (g per 100 mL, solvent). Melting points were

determined using a STUART Scientific, model SMP1, and are

not corrected. Solvents were removed in a B u¨ chi rotavapor.

s

General protocols

lipophilicity and BBB permeation with emphasis on conjugates

t

2

(A.1) Glutamate mono-functionalization. To a solution of

the appropriate glutamate mono methyl ester (1 equiv.) in

IVb and Vb (clog P of 1.61 and 1.30, PSA 108.05 A , BB ratio:

.655 and 0.739, respectively). Overall, this methodology

0

anhydrous CH

sequentially added Et

2

Cl

2

(25 mL) in a round-bottom flask was sub-

N (3 equiv.), TBTU (1.1 equiv.) and the

constitutes a great advance for the design and assembly of

s

3

Glypromate conjugates, paving the way for the discovery of

appropriate bioactive amine (1.2 equiv.). The resulting solution

was stirred for 2 h at RT under an inert atmosphere (Ar). After

reaction completion, the solvent was removed using a rotatory

evaporator. The residue obtained was dissolved in EtOAc

novel multitarget neuroprotective drugs.

Additionally, the formation of chiral glutarimides 6(a-c)

mediated by TBTU during the bis-functionalization of gluta-

mate is unprecedented, oﬀering an alternative route for the

preparation of glutarimide alkaloids such as julocrotine and its

derivatives.

(

100 mL), transferred into a separatory funnel and washed with

saturated aqueous solution of NaHCO

(3 ꢁ 100 mL). The

organic phase was dried with anhydrous Na SO , filtered and

3

2

4

concentrated. The resulting residue was purified by chromato-

graphic column using an appropriated eluent, specified for each

compound.

Experimental section

Chemistry. General data

(

A.2) Glutamate bis-functionalization and preparation of

glutarimides. To a solution of Boc-Glu(OH)-OH (1 equiv.)

in anhydrous CH Cl (25 mL) in a round-bottom flask was

subsequentially added Et N (4 equiv.), TBTU (2.2 equiv.) and

All chemicals were of reagent grade and were used without

further purifications: Boc-Glu(OH)-OMe, Boc-Glu(OMe)-OH,

Boc-Gly-OSu, amantadine and triethylamine were obtained

from Fluorochem; TBTU was obtained from Bachem; H-Pro-OH,

Boc-Pro-OH and Boc-Glu(OH)-OH was obtained from Merk;

2

3

the appropriate bioactive amine (2.2 equiv.). The resulting

solution was stirred for 2 h at RT under an inert atmo-

sphere (Ar). Work-up and purification follow the same protocol

described in A.1.

(R)-1-aminoindane was obtained from Alfa Aesar; memantine

was obtained from Acros Organics; formaldehyde was obtained

from Fisher Scientific; sodium triacetoxyborohydride was obtained

from Sigma-Aldrich and trifluoroacetic acid was obtained from

VWR. All air sensitive reactions were carried out under argon

atmosphere. Analytical TLC was carried out on pre-coated silica gel

plates (Merck 60 F254, 0.25 mm) using UV light and an ethanolic

solution of phosphomolybdic acid (followed by gentle heating) for

visualization. Flash chromatography was performed on silica gel

(B) One-pot assembly of conjugates. To a solution of

L-proline (1 equiv.) in anhydrous CH Cl (25 mL) in a round-

2

bottom flask was added Boc-Gly-OSu (1.1 equiv.), and the

reaction was left stirring for 12 h at RT under inert atmosphere

(

Ar). Next, to this solution was subsequentially added TBTU

1.1 equiv.) and, after 30 min, the appropriate functionalized

glutamate (1.2 equiv.). Work-up and purification follow the

same protocol described in A.1.

(Merck 60, 230–240 mesh).

(

C) Removal of N-Boc protecting group. To a solution of

N-Boc protected compound (1 equiv.) in anhydrous CH Cl

H- and C-NMR spectra were recorded at Centro de Materiais (30 mL) in a round-bottom flask was added TFA (30 equiv.),

da Universidade do Porto (CEMUP) with a Bruker Avance III 400 and the reaction was left stirring for 1 h at RT under inert

Apparatus

2

1

13

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Paper

NJC

atmosphere (Ar). After that, the volatiles were removed using a

rotatory evaporator.

C

E

-1 + C ); 53.7

E 3 2 3

(Cq, C -1); 53.3 (CH, C -2); 52.5 (CH , CO CH ); [50.7, 47.6,

E

-5]; 155.9 (Cq, OCONH); 80.1 (Cq, C(CH

3

)

3

ꢀ

c

(

D) Reductive amination. To a solution of the conjugate 42.8, 40.2 (CH ), C -2 + C -4 + C -6 + C -8 + C -9 + C -10 + C -3 +

2 c c c c c c E

ammonium salt (0.6139 mmol) in DCE (30 mL) in a round C -4]; 32.5 (2 ꢁ Cq, C -3 + C -7); [30.2, 28.5, 21.2, (CH +

E

c

bottom flask was subsequentially added CH O (37% m/v, 5 ꢁ CH ), C -11 + C -12 + C(CH ) + C -5]. ESI-MS m/z:

2

3

c

ꢀ

3 3

c

+

1

.54 mmol) and NaBH(OAc)

suspension was stirred for 2 h. The solvent was then removed

using a rotatory evaporator and CH Cl (25 mL) was added protocol A.1, starting from 0.4922 g of Boc-Glu(OMe)-OH. The

3

(3.069 mmol). The resulting [M + H] calcd for [C23

H

39

N

2

O

5

] 423.29, found 423.26.

Synthesis of Boc-Glu(OMe)-aminoindane (3a). General

2

to induce the precipitation of excess reductive agent and resulting residue was purified by chromatographic column

inorganic salts followed by filtration. The filtrate was concen- using EtOAc. Aspect: light brown solid. Yield: 91% (0.6454 g).

1

9

trated in vacuo providing in most cases the N,N-dimethylated M.p.: 96–98 1C. [a] = ꢀ24.0 ꢂ 0.1 (c1, CHCl ). Analytical data:

D

3

1

conjugates pure enough to be analytically characterized.

H-NMR (CDCl , 400 MHz) d ppm: 7.26–7.12 (m, 4H, H -4 +

3 a

Synthesis of Boc-Glu(aminoindane)-OMe (1a). General H -5 + H -6 + H -7); 6.56 (d, J = 6.8 Hz, 1H, CONH); 5.43 (q,

a

protocol A.1, starting from 0.4909 g of Boc-Glu(OH)-OMe. The J = 7.8 Hz, 1H, H

resulting residue was purified by chromatographic column -2); 3.66 (s, 3H, CO

using EtOAc. Aspect: brown solid. Yield: 95% (0.6720 g). M.p.: (m, 3H); 2.15 (dtd, J = 12.7, 7.3, 5.5 Hz, 1H), H

a

-1); 5.35 (d, J = 8.0 Hz, 1H, CONH); 4.15 (s, 1H,

CH ); 3.02–2.77 (m, 2H, H -3); [2.64–2.35

-2 + H -3]; 1.99–

). C-NMR/DEPT-135

, 101 MHz) d ppm: [173.9 (Cq), 171.4 (Cq), C -1 + C -5];

H -5 + H -6 + H -7); 6.23 (d, J = 6.9 Hz, 1H, CONH); 5.43 (q, 155.8 (Cq, CONH); [143.4 (Cq), 142.9 (Cq), C -3a + C -7a]; [128.1

H

E

2

3

a

E

1

8

13

1

20–122 1C. [a]

D

= ꢀ655.4 ꢂ 0.1 (c1, CHCl

3

). Analytical data: 1.73 (m, 2H, H

E 3 3

-4); 1.41 (s, 9H, C(CH )

1

H-NMR (CDCl

3

a

, 400 MHz) d ppm: 7.30–7.14 (m, 4H, H -4 + (CDCl

3

E

a

J = 7.7 Hz, 1H, H -1); 5.34 (d, J = 7.5 Hz, 1H, CONH); 4.27 (br s, (CH), 126.9 (CH), 124.9 (CH), 124.1 (CH), C -4 + C -5 + C -6 + C -7];

a

1

H, H -2); 3.72 (s, 3H, CO CH ); 3.00–2.81 (m, 2H, H -3); 2.56 80.2 (Cq, C(CH ) ); [54.7 (CH), 53.9 (CH), C -1 + C -2]; 51.9 (CH ,

E

2

3

a

ꢀ

3 3

a

E

3

(

dtd, J = 12.8, 7.9, 4.0 Hz, 1H), 2.36–2.16 (m, 3H); 2.00–1.73 CO

2

CH

3

); [34.1 (CH

-3 + C

-4]; 27.9 (3 ꢁ CH

2

), 30.4 (CH

2

), 30.3 (CH

2

), 28.4 (CH

2

), C

a

-2 +

1

3

m, 2H); 1.40 (s, 9H, C(CH

01 MHz) d ppm: [172.9 (Cq), 171.8 (Cq), C

Cq, OCONH); [143.5 (Cq), 143.2 (Cq), C -3a + C

CH), 126.8 (CH), 124.9 (CH), 124.2 (CH), C -4 + C

3

)

3

). C-NMR/DEPT-135 (CDCl

-1 + C -5]; 155.8 [M + H] calcd for [C20

-7a]; [128.0 Synthesis of Boc-Glu(OMe)-amantadine (3b). General

-5 + C -6 + protocol A.1, starting from 0.5803 g of Boc-Glu(OMe)-OH. The

3

,

C

a

-3 + C

E

3

, C(CH

3 3

) ). ESI-MS m/z:

ꢀ

+

1

(

E

29 2 5

H N O ] 377.21, found 377.24.

a

C -7]; 80.2 (Cq, C(CH ) ); [54.9 (CH), 53.2 (CH), C -1 + C -2]; resulting residue was purified by chromatographic column

a

ꢀ

3 3

a

E

5

(

2.5 (CH ,CO CH ); [34.0 (CH ), 32.8 (CH ), 30.3 (CH ), 28.9 using EtOAc. Aspect: colourless oil. Yield: 89% (0.7798 g).

3 2 3 2 2 2

1

8

1

CH ), C -2 + C -3 + C -3 + C -4]; 28.4 (3 ꢁ CH , C(CH ) ). ESI-MS [a] = ꢀ14.3 ꢂ 0.2 (c1, CHCl ). Analytical data: H-NMR (CDCl ,

ꢀ

+ +

2

a

E

3

3 3

D

3

m/z: [M + H] calcd for [C20

H

29

N

2

O

5

] 377.21, found 377.22.

400 MHz) d ppm: 5.88 (br s, 1H, CONH); 5.26 (d, J = 7.7 Hz, 1H,

-2); 3.67 (s, 3H, CO CH ); 2.54–2.27

-4); 2.11–1.80 (m, 11H, H -3 + H -2 + H -3); 1.66

). C-NMR/DEPT-135 (CDCl

-1 + C -5]; 155.8

+14.5 ꢂ 0.1 (c1, CHCl ). Analytical data: H-NMR (CDCl , (Cq, OCONH); 79.9 (Cq, C(CH ) ); 54.1 (CH, C -2); 51.9

Synthesis of Boc-Glu(amantadine)-OMe (1b). General CONH); 3.99 (s, 1H, H

E

2

3

protocol A.1, starting from 0.5821 g of Boc-Glu(OH)-OMe. The (m, 2H, H

resulting residue was purified by precipitation using Et O. (t, 6H, H

E

b

1

3

2

b

-4); 1.42 (s, 9H, C(CH

3

)

3

,

Aspect: white solid. Yield: 90% (0.7911 g). M.p.: 190–191 1C. [a] 101 MHz) d ppm: [173.9 (Cq), 170.4 (Cq), C

E

1

D

6

1

=

3

ꢀ

b

3 3

E

4

00 MHz) d ppm: 5.53 (br s, 1H, CONH); 5.27 (d, J = 6.5 Hz, 1H, (CH , CO CH ); 52.2 (Cq, C -1); [41.6, 36.4, 30.4, (6 ꢁ CH ),

3

2

3

2

CONH); 4.25 (m, 1H, H -2); 3.72 (s, 3H, CO CH ); [2.23–1.84 C -2 + C -4]; 29.5 (3 ꢁ CH, C -3); 28.5 (2 ꢁ CH , C -3 + C -4);

E

2

3

b

2

E

+

(

[

(

m, 15H), 1.66 (s, 4H), H

E

-3 + H

). C-NMR/DEPT-135 (CDCl

173.0 (Cq), 171.1 (Cq), C -1 + C -5]; 155.8 (Cq, OCONH); 80.1

Cq, C(CH ); 53.3 (CH, C -2), 52.5 (CH , CO CH ); 52.1 A.1, starting from 0.4021 g of Boc-Glu(OMe)-OH. The resulting

-1); 41.7 (3 ꢁ CH , C -2), 36.5 (3 ꢁ CH , C -3), 33.8, residue was purified by chromatographic column using EtOAc.

CH , C -4), 29.5 (3 ꢁ CH, C -4), 28.8 (CH , C -3); 28.4 (3 ꢁ CH , Aspect: white solid with low melting point. Yield: 89% (0.5788 g).

E

-4 + H

b

-2 + H

b

-3 + H

b

-10]; 1.42 28.4 (3 ꢁ CH

3

, C(CH

3

)

3

). ESI-MS m/z: [M + H] calcd for

] 395.25, found 395.25.

Synthesis of Boc-Glu(OMe)-memantine (3c). General protocol

ꢀ

1

3

+

s, 9H, C(CH

3

)

3

3 35 2 5

, 101 MHz) d ppm: [C21H N O

E

3

)

3

E

3

2

3

ꢀ

Cq, C

b

2

b

2

b

2

E

b

2

E

3

+

22

1

C(CH ) ). ESI-MS m/z: [M + H] calcd for [C H N O ] 395.25, [a]_D= ꢀ16.5 ꢂ 0.3 (c1, CHCl ). Analytical data: H-NMR (CDCl ,

ꢀ

3 3

21 35

2

5

3

found 395.23.

Synthesis of Boc-Glu(memantine)-OMe (1c). General CONH); 4.00 (br s, 1H, H

protocol A.1, starting from 0.4013 g of Boc-Glu(OH)-OMe. The (m, 2H, H -4); [1.94–1.75 (m, 4H), 1.70–1.56 (m, 4H), 1.51–1.30

resulting residue was purified by chromatographic column (m, 13H), 1.22–1.08 (m, 3H), H -3 + C(CH + H -2 + H -3 + H -4 +

-8 + H -9 + H -10]; 0.84 (s, 6H, H -11 +

-12). C-NMR/DEPT-135 (CDCl , 101 MHz) d ppm: [173.9 (Cq),

00 MHz) d ppm: 5.57 (br s, 1H, CONH); 5.29 (d, J = 7.0 Hz, 170.5 (Cq), C -1 + C -5]; 155.8 (Cq, OCONH); 80.0 (Cq, C(CH ) );

400 MHz) d ppm: 5.94 (br s, 1H, CONH); 5.26 (d, J = 7.9 Hz, 1H,

E

-2); 3.68 (s, 3H, CO CH ); 2.59–2.32

2

3

E

3

)

3

c

2

D

2

using EtOAc. Aspect: yellow oil. Yield: 94% (0.6101 g). [a]

=

H

c

-5 + H

c

13

-6 + H

c

-7 + H

c

1

+

4

1

2

52.2 ꢂ 0.4 (c1, CHCl

3

). Analytical data: H-NMR (CDCl

3

,

c

3

E

ꢀ

3 3

H, CONH); 4.27 (br s, 1H, H -2); 3.74 (s, 3H, CO CH ); 2.22– 53.8 (Cq, C -1); 54.1 (CH, C -2); 51.9 (CH , CO CH ); [50.7, 47.6,

E

2

3

c

E

3

2

3

.11 (m, 4H, H -3 + H -4); [1.71–1.60 (m, 5H), 1.54–1.31 42.8, (6 ꢁ CH ), C -2 + C -4 + C -6 + C -8 + C -9 + C -10]; 40.2

E

2

c

(

m, 17H), C(CH

3

)

3

+ H

c

-2 + H

c

-3 + H -3 + C

-11 + H + C

39 2 5

, 101 MHz) d ppm: [173.0 (Cq), 171.1 (Cq), [M + H] calcd for [C23H N O ] 423.29, found 423.31.

c

-4 + H

c

-5 + H

c

-6 + H

c

-7 + (2 ꢁ CH

2

, C

E

-3 + C

E

-4); 32.5 (2 ꢁ Cq, C

c

-7); [30.2, 28.4, 21.2,

1

3

H

c

-8 + H

c

-9 + H

c

-10]; 0.85 (s, 6H, H

c

-12). C-NMR/DEPT- (CH + 5 ꢁ CH

3

), C

c

-11 + C -12 + C(CH

c

3

)

3

c

-5]. ESI-MS m/z:

ꢀ

+

1

35 (CDCl

3

2

1056 | New J. Chem., 2020, 44, 21049--21063

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Synthesis of Boc-Glu(aminoindane)-aminoindane (5a) and 28.5 (3 ꢁ CH

Paper

+

3

, C(CH

ꢀ

3

)

3

). ESI-MS m/z: [M + H] calcd for

glutarimide 6a. General protocol A.2, starting from 0.3460 g of [C30H N O ] 514.36, found 514.39. Analytical data for 6b:

48 3 4

+

2

0

1

Boc-Glu(OH)-OH. The resulting residue was purified by chro- m.p.: 193–195 1C. [a] = ꢀ46.5 ꢂ 0.2 (c1, CHCl ). H-NMR

D

3

matographic column using EtOAc obtaining 0.2340 g (35% (CDCl , 400 MHz) d ppm: 5.61 (br s, 1H, CONH), 4.36 (dd,

3

yield) of a beige solid identified as 5a and 0.1450 g (30% yield) J = 8.9, 2.6 Hz, 1H, H -2), 2.73 (dt, J = 17.6, 9.9 Hz, 1H), 2.43

E

of a beige solid identified as 6a. Analytical data for 5a: m.p.: (ddd, J = 17.5, 9.4, 3.3 Hz, 1H), 2.20 (ddd, J = 19.1, 12.9, 9.4 Hz,

2

0

1

4

H

[

95–196 1C. [a]

D

= +116.6 ꢂ 0.4 (c1, CHCl

3

). H-NMR (CDCl

3

,

1H), 2.14–1.92 (m, 10H), 1.77–1.61 (m, 6H), 1.52 (s, 9H,

1

3

00 MHz), rotamers present (70 : 30), d ppm: 7.33–7.09 (m, 8H, C(CH

3

)

3

). C-NMR/DEPT-135 (CDCl

-7 ); (Cq), 169.7 (Cq), C -1 + C -5]; 150.0 (Cq, OCONH); 83.7 (Cq,

3 E 2 3 b

); [60.8 (CH + CH ), C -2 + CO CH ]; 52.3 (Cq, C -1);

3

, 101 MHz) d ppm: [174.1

ꢀ

0

-4 + H

a

-5 + H

a

-6 + H

a

-7 + H

a

0

-4 + H

a

0

-5 + H

a

0

-6 + H

a

0

E

a

ꢀ

6.94 (d, J = 6.0 Hz, minor), 6.80 (d, J = 8.3 Hz, major), 1H, C(CH

3

)

3

ꢀ

CONH]; [6.30 (d, J = 7.0 Hz, major), 6.25 (d, J = 8.3 Hz, minor), [41.6, 36.4, 31.7, 22.4 (6 ꢁ CH ), C -2 + C -4];, [29.5, 28.1 (3 ꢁ

2

b

+

1

H, CONH]; [5.87 (d, J = 4.8 Hz, minor), 5.76 (d, J = 7.3 Hz, CH, C -3) + (3 ꢁ CH , C(CH ) )]. ESI-MS m/z: [M + H] calcd for

c

3

ꢀ

3 3

0

+

major), 1H, CONH]; 5.49–5.34 (m, 2H, H -1 + H

0

-1 ); 4.21–4.07 [C H N O ] 363.23, found 363.28.

a

20 31 2 4

0

(m, 1H, H

E

-2); 2.96 (ddd, J = 15.6, 8.6, 3.7 Hz, 2H, H

a

-3 + H

-3 ); 2.60–2.48 (m, glutarimide 6c. General protocol A.2, starting from 0.7398 g

H); 2.43–2.28 (m, 2H); 2.21–1.93 (m, 2H); 1.86–1.73 (m, 2H); of Boc-Glu(OH)-OH. The resulting residue was purified by

a

0

-3 );

Synthesis of Boc-Glu(memantine)-memantine (5c) and

0

2

1

.84 (dt, J = 16.0, 8.0 Hz, 2H, 2 ꢁ H

a a

-3 + H

0

1

3

3 3

.43–1.39 [1.41 (s), 1.40 (s), 9H, C(CH ) ]. C-NMR/DEPT-135 chromatographic column using Hex/EtOAc (1 : 2) obtaining

(CDCl

3

, 101 MHz) d ppm: [143.4 (Cq), 143.4 (Cq), 143.3 (Cq), 0.4271 g (25% yield) of a yellow oil identified as 5c and

1

C_a

43.1 (Cq), 142.9 (Cq), C -1 + C -5 + CONH + C -3a + C -7a + 0.2949 g (19% yield) of a colourless oil identified as 6c.

E

a

0

23

1

0

-3a + C_a

0

-7a ]; [128.1 (CH), 126.8 (CH), 124.9 (CH), 124.2 Analytical data for 5c: [a]_D= ꢀ5.6 ꢂ 0.1 (c1, CHCl ). H-NMR

3

0

(CH), C -4 + C -5 + C -6 + C -7 + C

0

-4 + C

0

-5 + C

0

-6 + C

0

-7 ]; (CDCl , 400 MHz) d ppm: 6.20 (d, J = 6.0 Hz, 1H, CONH), 5.60

a

3

0

80.2 (Cq, C(CH

3

)

3

); [54.9, 54.7, (3 ꢁ CH), C

a

-1 + C

a

0

-1 + C

E

-2]; (d, J = 7.5 Hz, 2H, 2 ꢁ CONH), 3.95 (d, 1H, H

E

-2), 2.30–2.09

ꢀ

2

[33.9 (CH

), 32.9 (CH

-3 + C

2

), 30.4 (CH

2

), 30.3 (CH

2

), C

a

-2 + C

a

-3 + (m, 4H, H -3 + H -4), 2.01–1.79 (m, 6H), 1.63 (s, 8H), 1.44 (s, 9H,

E

0

C

a

0

-2 + C

a

0

E

-3 + C

E

-4]; 28.4 (3 ꢁ CH

3

, C(CH

3

)

3

). ESI-MS C(CH

3

)

3

), 1.39–1.33 (m, 4H), 1.30–1.24 (m, 4H), 1.18–1.08

ꢀ

+

0

m/z: [M + H] calcd for [C28

Analytical data for 6a: m.p.: 200–202 1C. [a]

H

36

N

3

O

4

]

478.27, found 478.30. (m 4H), 0.83 (d, J = 2.5 Hz, 12H, H

c

-11 + H

c

-12 + H

c

0

-11 +

2

0

13

D

= +58.1 ꢂ 0.2

H -12 ). C-NMR/DEPT-135 (CDCl , 101 MHz) d ppm: [172.1

c

0

3

1

(

c1, CHCl₃). H-NMR (CDCl , 400 MHz) d ppm: 7.27–7.10 (Cq), 170.9 (Cq), C -1 + C -5]; 155.9 (Cq, OCONH); 79.8 (Cq,

m, 4H, H -4 + H -5 + H -6 + H -7); 6.61 (d, J = 8.0 Hz, 1H, C(CH ) ); 53.8 (CH, C -2); 53.7 (CH , CO CH ); [50.7, 47.6, 42.8,

3

E

ꢀ

a

ꢀ

3 3

E

3

2

3

0

-2 +

c

CONH); 5.44 (q, J = 7.7 Hz, 1H, H -1); 4.51 (dd, J = 8.9, 2.9 Hz, 40.2, (12 ꢁ CH ), C -2 + C -4 + C -6 + C -8 + C -9 + C -10 + C

0

a

2

c

0

c

0

1

8

H, H

.1 Hz, 1H, H

E

-2); 2.98 (ddd, J = 15.8, 8.7 Hz, 1H, H

a

-3); 2.86 (dt, J = 16.0,

C

c

0

-4 + C

c

0

-6 + C

-3 + C

C(CH

c

0

-8 + C

c

0

-9 + C

-7 + C

c

0

-10 ]; 32.5 (Cq, C

c

-1 + C

c

0

-1 ),

0

-3); 2.69 (dt, J = 17.5, 9.8 Hz, 1H); 2.55 (dtd, 30.2 (6 ꢁ CH, C

c

-5 + C

c

0

-3 + C

c

0

-5 + C

c

0

-7 ); 28.5

a

+

J = 11.8, 7.9, 4.0 Hz, 1H); 2.40 (ddd, J = 17.4, 9.4, 3.4 Hz, 1H); (3 ꢁ CH

3

,

3

)

3

). ESI-MS m/z: [M + H] calcd for

ꢀ

+

2.23 (ddd, J = 18.7, 13.0, 9.5 Hz, 1H); 2.11 (ddt, J = 12.9, 9.7, 3.1 [C34H N O ] 570.43, found 570.50. Analytical data for 6c:

56 3 4

2

3

1

Hz, 1H); 1.83 (ddd, J = 16.0, 12.9, 8.4 Hz, 1H); 1.47 (s, 9H, [a]

C(CH ) ). C-NMR/DEPT-135 (CDCl , 101 MHz) d ppm: [174.0 d ppm: 5.55 (br s, 1H, CONH), 4.28 (dd, J = 8.8, 2.7 Hz, 1H,

D

= ꢀ17.9 ꢂ 0.2 (c1, CHCl

3 3

). H-NMR (CDCl , 400 MHz)

1

3

(

Cq), 170.8 (Cq), 149.9 (Cq), 143.2 (Cq), 142.8 (Cq), C -1 + C -5 + H -2), 2.20–2.00 (m, 4H, H -3 + H -4), 1.75 (d, J = 2.9 Hz, 3H),

E E E E E

OCONH + C -3a + C -7a]; [128.1 (CH), 126.8 (CH), 124.8 (CH), 124.2 1.65–1.53 (m, 4H), 1.46 (s, 9H, C(CH ) ), 1.37 (d, J = 2.0 Hz, 2H),

a

3 3

(

CH), C

2 ꢁ CH), C

CH ), C -2 + C

m/z: [M + H] calcd for [C19

Synthesis of Boc-Glu(amantadine)-amantadine (5b) and 47.7, 42.7, 40.2, 30.2, 22.3 (6 ꢁ CH

glutarimide 6b. General protocol A.2, starting from 0.8230 g C -9 + C -10]; 32.5 (Cq, C -1), 30.1 (3 ꢁ CH, C -3 + C -5 + C -7);

a

-4 + C

-1 + C

-3 + C

a

-5 + C

-2]; [33.6 (CH

-3 + C

-4]; 27.9 (3 ꢁ CH

a a 3 3

-6 + C -7]; 83.7 (Cq, C(CH ) ); [60.2, 54.8, 1.31 (d, J = 12.1 Hz, 2H), 1.26–1.16 (m, 2H), 0.78 (d, J = 1.9 Hz,

ꢀ

1

3

a

E

2

), 31.6 (CH

2

), 30.2 (CH

, C(CH ). ESI-MS ppm: [174.1 (Cq), 169.9 (Cq), C

] 345.18, found 345.19. 83.8 (Cq, C(CH ); [60.8, (CH + CH

), C

2

), 22.3 6H, H

c

-11 + H

c

-12). C-NMR/DEPT-135 (CDCl

-1 + C -5]; 150.2 (Cq, OCONH);

), C

-2 + C

3

, 101 MHz) d

2

a

E

3

ꢀ

3

)

3

E

ꢀ

+

H

25

N

2

O

4

3

)

3

E

-2 + CO

2

CH

3

]; [50.6,

-8 +

ꢀ

2

c

-4 + C

c

-6 + C

c

of Boc-Glu(OH)-OH. The resulting residue was purified by 28.43 (2 ꢁ CH , C -11 + C -12); 28.1 (3 ꢁ CH , C(CH ) ). ESI-MS

3

c

3

ꢀ

3 3

+

chromatographic column using EtOAc obtaining 0.5651 g m/z: [M + H] calcd for [C H N O ] 391.26, found 391.23.

2

35 2 4

(33% yield) of a white solid identified as 5b and 0.3262 g

Synthesis of H-Glu(aminoindane)-OMe (2a). General proto-

(

27% yield) of a white solid identified as 6b. Analytical data col C, starting from 0.6441 g of 1a. Aspect: brown oil. Yield: 97%

2

0

1

17

for 5b: m.p.: 42–45 1C. [a]

D

= +56.8 ꢂ 0.4 (c1, CHC

3

). H-NMR (0.6479 g). [a]

D

= ꢀ35.6 ꢂ 0.2 (c1, CHCl

OD, 400 MHz) d ppm: 7.28–7.14 (m, 4H, H

-6 + H -7); 5.37 (t, J = 7.5 Hz, 1H, H -1); 4.15–4.09 (t,

H -2); 2.31–2.13 (m, 2H, H -4); 2.12–2.03 (m, 6H); 2.03–1.96 J = 6.3 Hz, 1H, H -2); 3.85 (s, 3H, CO CH ); 3.01 (m, 1H, H -3);

3

). Analytical data:

1

(CDCl

3

, 400 MHz) d ppm: 6.16 (br s, 1H, CONH); 5.59 (d, J =

H-NMR (CD

-5 + H

3

a

-4 +

7

.3 Hz, 1H, CONH); 5.50 (br s, 1H, CONH); 4.05–3.87 (m, 1H,

H

a

E

2

3

a

(

m, 12H); 1.95–1.82 (m, 2H, H -3); 1.73–1.61 (m, 12H); 1.44 2.85 (m, 1H, H -3); [2.58–2.42 (m, 3H), 2.22 (dp, J = 14.6, 6.9 Hz,

E a

1

3

s, 9H, C(CH ) ). C-NMR/DEPT-135 (CDCl , 101 MHz) d ppm: 2H), 1.85 (ddd, J = 16.0, 12.8, 8.2 Hz, 1H), H -2 + H -3 + H -4].

3

a

E

1

3

[

171.9 (Cq), 170.7 (Cq), C

CH , CO CH ); 52.2 (Cq, C

-2 + C -4 + C

E

-1 + C

E

-5]; 52.2 (CH, C

-1); [41.7, 41.6, 36.5, (CH

-4 ]; 29.6 (6 ꢁ CH, C

E

-2); 52.1

C-NMR/DEPT-135 (CD

), 170.7 (Cq), C -1 + C -5]; [144.6 (Cq), 144.2 (Cq), C

-9]; [128.9 (CH), 127.7 (CH), 125.7 (CH), 124.9 (CH),

3

OD, 101 MHz) d ppm: [173.8 (Cq),

(

C

3

2

3

b

2

E

a

-4 +

0

-2 + C

0

-3 + C

0

-3 );

C

a

b

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New J. Chem., 2020, 44, 21049--21063 | 21057

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Paper

NJC

C

(

a

-4 + C

CH , CO

C -2 + C -3 + C -3 + C -4]. ESI-MS m/z: [M] calcd for (0.5292 g). [a] = +7.7 ꢂ 0.9 (c1, CHCl ). Analytical data:

a

-5 + C

a

-6 + C

); [34.3 (CH

a

-7]; [55.9 (CH), 53.7 (CH), C

a

-1 + C

E

-2]; 53.6

Synthesis of H-Glu(OMe)-memantine (4c). General protocol

3

2

CH

3

2

), 32.3 (CH ), 31.0 (CH

2

), 27.2 (CH

2

), C, starting from 0.5281 g of 3c. Aspect: brown oil. Yield: 97%

+

21

D

a

E

3

+

1

[C H N O ] 277.15, found 277.14.

H-NMR (CDCl , 400 MHz) d ppm: 6.90 (br s, 1H, CONH);

3

1

5

21

2

3

Synthesis of H-Glu(amantadine)-OMe (2b). General protocol 4.12 (d, J = 7.1 Hz, 1H, H -2); 3.70 (s, 3H, CO CH ); [2.56 (q,

E

2

3

C, starting from 0.7571 g of 1b. Aspect: yellow oil. Yield: 98% J = 18.2 Hz, 2H), 2.13 (s, 3H), 1.79 (s, 2H), 1.72–1.48 (m, 4H),

2

(

0.7681 g). [a]

D

= +25.6 ꢂ 0.1 (c1, CHCl

3

). Analytical data: 1.40–1.24 (m, 5H), 1.14 (s, 2H), H

-5 + H -6 + H -8 + H -9 + H -10]; 0.84 (s, 6H, H

C-NMR/DEPT-135 (CDCl , 101 MHz) d ppm: [174.5 (Cq), 167.3

-4). C-NMR/ (Cq), C -1 + C -5]; 54.9 (Cq, C -1); 53.4 (CH, C -2); 52.6 (CH

E

-3 + H

E

-4 + H

c

-2 + H

c

-4 +

-12).

1

H-NMR (CD

-2); 3.84 (s, 3H, CO

.21 (m, 11H, H -3 + H

3

OD, 400 MHz) d ppm: 4.06 (t, J = 6.4 Hz, 1H,

H

c

-11 + H

c

1

3

H

2

E

2

CH

3

); 2.39 (dd, J = 10.5, 4.3 Hz, 2H, H

E

-4);

3

1

3

E

b

-2 + H

b

-3); 1.71 (s, 6H, H

b

E

c

E

3

,

DEPT-135 (CD OD, 101 MHz) d ppm: [173.2 (Cq), 170.7 (Cq), CO CH ); [50.5, 47.1, 42.6, (6 ꢁ CH ), C -2 + C -4 + C -6 + C -8 +

3

2

3

2

c

C -1 + C -5]; 53.7 (CH, C -2); 53.7 (CH , CO CH ); 53.0 (Cq, C -9 + C -10]; 39.7 (2 ꢁ CH , C -3 + C -4); 32.5 (2 ꢁ Cq, C -3 +

E

3

2

3

c

2

E

c

C -1); [42.3, 37.5, 33.2 (6 ꢁ CH ), C -2 + C -4]; 30.9 (3 ꢁ CH, C -7); [30.0, 26.8, (CH + 2 ꢁ CH ), C -5 + C -11 + C -12]. ESI-MS

b

2

b

c

3

c

+

C

b

-3 + C

b

-5 + C

b

-7); 27.3 (2 ꢁ CH

2

, C

E

-3, C

E

-4). ESI-MS m/z: [M]

m/z: [M] calcd for [C18

31 2 3

H N O ] 323.23, found 323.28.

Synthesis of Boc-Gly-Pro-Glu(aminoindane)-OMe (7a).

+

27 2 3

calcd for [C16H N O ] 295.20, found 295.18.

Synthesis of H-Glu(memantine)-OMe (2c). General protocol General protocol B, starting from 0.1521 g of L-proline. The

C, starting from 0.5590 g of 1c. Aspect: yellow oil. Yield: 98% resulting residue was purified by chromatographic column

2

1

(

0.5659 g). [a]

D

= ꢀ8.2 ꢂ 0.9 (c1, CHCl

3

). Analytical data: using EtOAc. Aspect: light brown solid. Yield: 70% (0.4907 g).

1

15

H-NMR (CDCl , 400 MHz) d ppm: 6.08 (br s, 1H, CONH); M.p.: 43–45 1C. [a] = ꢀ22.0 ꢂ 0.3 (c1, CHCl ). Analytical data:

3

D

3

1

4

.14 (d, J = 7.1 Hz, 1H, H -2); 3.82 (s, 3H, CO CH ); [2.54–2.14

H-NMR (CD OD, 400 MHz), rotamers present (85 : 15), d ppm:

3

E

2

3

(

m, 5H), 1.79 (s, 2H), 1.58 (q, J = 12.0 Hz, 5H), 1.38–1.25 (m, 5H), 7.34–7.11 (m, 4H, H -4 + H -5 + H -6 + H -7); 5.37 (t, J = 7.8 Hz,

a

1

H

(

5

4

.14 (s, 2H), H

-9 + H -10]; 0.85 (s, 6H, H

CDCl , 101 MHz) d ppm: [172.5 (Cq), 169.7 (Cq), C

4.7 (Cq, C -1); 53.7 (CH, C -2); 52.9 (CH , CO CH ); [50.6, 47.2,

2.6, (6 ꢁ CH ), C -2 + C -4 + C -6 + C -8 + C -9 + C

E

-3 + H

E

-4 + H

c

-2 + H

-11 + H

c

-4 + H

c

-5 + H

c

-6 + H

c

-8 + 1H, H

a

-1); 4.53–4.30 (m, 2H, H

E

-2 + H

P

-2); 3.85 (dd, J = 16.9,

1

3

c

-12). C-NMR/DEPT-135 3.9 Hz, 1H, H

G

-2); 3.73 (s, 3H, CO

-5); 3.00–2.75 (m, 2H, H -3); 2.56–2.28 (m, 4H, H

-2); 2.24–1.78 (m, 6H, H -4 + H -3 + H -4); [1.41 (s, major),

]. C-NMR/DEPT-135 (CD OD,

2

CH

3

); 3.58–3.46 (m, 3H,

3

E

-1 + C

E

-5];

H

G

-2 + 2H

P

a

P

-3 +

c

E

3

2

3

a

P

E

3

E

1

2

c

-10]; 39.7 1.36 (s, minor), 9H, C(CH

3

)

3

(

2 ꢁ CH , C -3 + C -4); 32.5 (2 ꢁ Cq, C -3 + C -7); [30.1, 26.1, 101 MHz), rotamers present, d ppm: [174.5 (Cq), 174.4 (Cq),

2

E

c

+

CH + 2 ꢁ CH ), C -5 + C -11 + C -12]. ESI-MS m/z: [M] calcd for 173.6 (Cq), 170.0 (Cq), C -1 + C -5 + C -1 + C -1]; 158.0 (Cq,

3

c

E

P

G

+

[C H N O ] 323.23, found 323.27.

CONH); [144.5 (Cq), 144.2 (Cq), C -3a + C -7a]; [128.8 (CH),

a a

1

8

31

2

3

Synthesis of H-Glu(OMe)-aminoindane (4a). General proto- 127.7 (CH), 125.7 (CH), 125.4 (CH), C

col C, starting from 0.6173 g of 3a. Aspect: brown oil. Yield: 98% 80.6 (Cq, C(CH

); [61.5, 55.8, 52.8, 52.7, (3 ꢁ CH + CH

-2 + CO CH ]; [47.7 (CH ), 43.7 (CH ), C -5 + C

-4 + 38.9 (CH, C -2); [34.7 (CH ), 32.9 (CH ), C -2 + C -3]; 31.0 (CH

-1); 3.91 (t, J = -4); 30.3 (CH , C , C(CH ]; [28.5 (CH

-4); [28.7, 28.6, 3 ꢁ CH

.5 Hz, 1H, H -2); 3.70 (s, 3H, CO CH ); 3.04 (ddd, J = 15.9, 8.7, 27.8 (CH ), C -3]; [25.9 (CH ), 23.4 (CH ), C -3]. ESI-MS m/z:

a

-4 + C

a

-5 + C

a

-6 + C

a

-7];

-1 +

-2];

)

3

), C

a

ꢀ

P

3

2

1

(

0.6274 g). [a]

D

= +60.9 ꢂ 0.3 (c1, CHCl

OD, 400 MHz) d ppm: 7.37–7.11 (m, 4H, H

-6 + H -7); 5.41 (t, J = 7.3 Hz, 1H, H

3

). Analytical data:

C

E

-2 + C

2

3

2

P

G

1

H-NMR (CD

-5 + H

3

a

P

2

a

2

,

H

a

C

P

2

E

3

)

3

2

),

ꢀ

P

6

4

2

E

2

3

2

E

2

+

.3 Hz, 1H, H -3); 2.90 (m, 1H, H -3); [2.62–2.41 (m, 3H); [M + H] calcd for [C H N O ] 531.28, found 531.32.

a

27 39 4 7

.30–2.07 (m, 2H); 1.97–1.83 (m, 1H), H -2 + H -3 + H -4].

Synthesis of Boc-Gly-Pro-Glu(amantadine)-OMe (7b). General

OD, 101 MHz) d ppm: [174.1 (Cq), protocol B, starting from 0.1671 g of L-proline. The resulting

-5]; [144.6 (Cq), 143.7 (Cq), C -3a + residue was purified by chromatographic column using EtOAc.

-7a]; [129.2 (CH), 127.7 (CH), 125.8 (CH), 125.2 (CH), C -4 + Aspect: white solid. Yield: 68% (0.5414 g). M.p.: 61–63 1C.

-5 + C -6 + C -7]; [56.1 (CH), 53.8 (CH), C -1 + C -2]; 52.5 (CH

CH ); [34.3 (CH ), 31.1 (CH ), 30.0 (CH ), 27.7 (CH ), C

a

E

1

3

C-NMR/DEPT-135 (CD

69.3 (Cq), C -1 + C

3

1

E

a

C

CO

a

1

6

1

a

E

3

,

[a]

D

= ꢀ9.8 ꢂ 0.3 (c1, CHCl

3 3

). Analytical data: H-NMR (CDCl ,

2

3

2

a

-2 + 400 MHz), rotamers present (85 : 15), d ppm: 7.23 (d, J = 7.6 Hz,

+

C -3 + C -3 + C -4]. ESI-MS m/z: [M] calcd for [C H N O ]

1H, CONH); [5.84 (br s, major), 5.73 (br s, minor), 1H, CONH];

a

E

15 21

2

3

277.15, found 277.19.

[5.48 (br s, minor), 5.40 (br s, major), 1H, CONH]; 4.56–4.41

Synthesis of H-Glu(OMe)-amantadine (4b). General protocol (m, 2H, H -2 + H -2); 4.05–3.83 (m, 2H, H -2); 3.72 (s, 3H,

E

P

G

C, starting from 0.7283 g of 3b. Aspect: colourless solid with CO

2

CH

3

); 3.63–3.34 (m, 2H, H

-3 + H -4 + H -2 + H

). C-NMR/DEPT-135 (CDCl

); 2.48 sent, d ppm: [172.5 (Cq), 171.4 (Cq), 171.3 (Cq), 168.4 (Cq),

-4); 2.16–2.01 (m, 11H, H -3 + -1 + C -5 + C -1 + C -1]; 155.9 (Cq, OCONH); 79.9 (Cq, C(CH );

H -2 + H -3); 1.73 (s, 6H, H -4). C-NMR/DEPT-135 (CD OD, [60.5, 52.5, 51.8, (2 ꢁ CH + CH ), C -2 + C -2 + CO CH ];

P

E

-5); 2.24–1.84 (m, 17H, H -3 +

2

low melting point. Yield: 98% (0.7390 g). [a]

D

= +5.4 ꢂ 0.1 (c1,

OD, 400 MHz) d ppm: C(CH

-2); 3.70 (s, 3H, CO CH

H

E

-4 + H

P

b

-3); 1.66 (s, 6H, H

b

-4); 1.44 (s, 9H,

1

13

CHCl

.80 (t, J = 6.4 Hz, 1H, H

td, J = 7.3, 1.3 Hz, 2H, H

3

). Analytical data: H-NMR (CD

3

)

3

, 101 MHz), rotamers pre-

3

E

2

3

(

E

C

E

P

G

3 3

)

ꢀ

1

3

b

3

P

E

2

3

1

01 MHz) d ppm: [174.2 (Cq), 168.3 (Cq), C -1 + C -5]; 53.9 52.0 (Cq, C -1); [47.3 (CH ), 46.5 (CH ), 43.3(CH ), C -2 +

E E b 2 2 2 G

(

3

C

CH, C -2); 53.5 (CH , CO CH ); 52.5 (Cq, C -1); [42.2, 37.3, C -5]; [41.5, 36.5 (6 ꢁ CH ), C -2 + C -4]; [33.2, 32.3, CH ,

E

3

2

3

b

P

2

b

2

0.8 (6 ꢁ CH

2

), C

b

-2 + C

b

-4]; 30.1 (3 ꢁ CH, C

b

-3); 27.8 (2 ꢁ CH

2

,

C

P

-4]; 29.5 (3 ꢁ CH, C

); 28.3 (CH , C -3); [24.9 (CH

m/z: [M + H] calcd for [C28

b

-3); 28.6 (CH

2

, C

E

-4); 28.5 (3 ꢁ CH

), C

45 4 7

H N O ] 549.33, found 549.30.

3

,

+

E

-3, C

E

-4). ESI-MS m/z: [M] calcd for [C16

H

27

N

2

O

3

] 295.20, C(CH

ꢀ

3

)

3

2

E

2

), 22.9 (CH

2

P

-3]. ESI-MS

+

found 295.16.

2

1058 | New J. Chem., 2020, 44, 21049--21063

This journal is ©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020

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NJC

Paper

Synthesis of Boc-Gly-Pro-Glu(memantine)-OMe (7c). General 168.8 (Cq), C

protocol B, starting from 0.1088 g of L-proline. The resulting (Cq, C(CH

E

-1 + C

E

-5 + C

P

-1 + C

G

-1]; 155.9 (Cq, CONH); 79.9

)

3 3

); [61.1, 53.3, 52.1, (2 ꢁ CH + CH

3 P E

), C -2 + C -2 +

ꢀ

residue was purified by chromatographic column using EtOAc. CO CH ]; 52.3 (Cq, C -1); [46.5 (CH ), 43.3 (CH ), C -2 + C -5];

2

3

b

2

G

P

1

8

Aspect: yellow solid with low m.p. Yield: 49% (0.2671 g). [a]

=

[41.5, 36.5, (6 ꢁ CH ), C -2 + C -4]; 30.3 (CH , C -4); 29.6 (3 ꢁ CH,

D

2

b

2

P

1

+

50.6 ꢂ 0.9 (c1, CHCl ). H-NMR (CDCl , 400 MHz), rotamers C -3); 28.9 (CH , C -4); 28.5 (3 ꢁ CH , C(CH ) ); 26.9 (CH , C -3);

3

b

2

E

3

ꢀ

3 3

2

E

+

present (80 : 20), d ppm: 7.69 (dd, J = 5.7, 3.3 Hz, 1H, CONH); 24.9 (CH

2 P 45 4 7

, C -3). ESI-MS m/z: [M + H] calcd for [C28H N O ]

[5.83 (s, major), 5.76 (s, minor), 1H, CONH]; [5.47 (s, minor), 549.33, found 549.35.

5

4

2

.37 (s, major), 1H, CONH]; 4.56–4.40 (m, 2H, H

.07–3.84 (m, 2H, H -2); 3.72 (s, 3H, CO CH ); 3.64–3.39 (m, protocol B, starting from 0.1089 g of L-proline. The resulting

H, H -5); 2.37–1.84 (m, 9H, H -3 + H -4 + H -3 + H -4 + H -5); residue was purified by chromatographic column using EtOAc.

E

-2 + H

P

-2);

Synthesis of Boc-Gly-Pro-Glu(OMe)-memantine (8c). General

G

2

3

P

E

c

2

4

[

1.83–1.59 (m, 3H), 1.51–1.07 (m, 18H), H -4 + H -6 + H -9, Aspect: brown oil. Yield: 51% (0.2782 g). [a] = ꢀ10.6 ꢂ 0.9

c

D

1

C(CH₃)₃+ H -2 + H -8 + H -10); 1.01–0.76 (m, 6H, H -11 + (c1, CHCl ). Analytical data: H-NMR (CDCl , 400 MHz), rota-

c

3

13

H -12)]. C-NMR/DEPT-135 (CDCl , 101 MHz), rotamers present, mers present (85 : 15), d ppm: 7.61 (d, J = 7.4 Hz, 1H, CONH);

c

3

d ppm: [172.3 (Cq), 171.8 (Cq), 167.4 (Cq), 167.0 (Cq), C

E

-1 + [6.40 (s, minor), 6.29 (s, major), 1H, CONH]; [5.41 (s, major),

); 5.30 (s, minor), 1H, CONH]; 4.53–4.19 (m, 2H, H -2 + H -2);

]; 4.08–3.84 (m, 2H, H -2); 3.63 (s, 3H, CO CH ); [2.54–2.32

-2 + (m, 1H), 2.81–2.62 (m, 1H), H -5]; 2.49–1.62 (m, 9H, H -3 +

-9 + -4 + H -3 + H -4 + H -5); 1.84–1.55 (m, 6H, H -4 + H -6 +

C

[

5

E

-5 + C

59.2, 58.7, 51.9, 51.8 (2 ꢁ CH + CH

1.4 (CH, C -5); 52.2 (Cq, C -1); [50.3 (CH

-5]; [42.3, 32.3, 29.8 (6 ꢁ CH ), C -2 + C -4 + C

P

-1 + C

G

-1]; 155.7 (Cq, OCONH); 79.2 (Cq, C(CH

3

)

3

E

P

ꢀ

3

), C

P

-2 + C

E

-2 + CO

2

CH

3

G

2

3

c

2

), 46.9 (CH

-6 + C -8 + C

2

), C

G

P

C

P

2

c

H

P

E

c

C -10]; 32.9 (2 ꢁ Cq, C -3 + C -7); [29.0 (CH ), 28.4 (CH ), C -4]; H -9); 1.43–1.03 (m, 15H, C(CH ) + H -2 + H -8 + H -10);

c

2

P

c

3 3

c

13

2

8.2 (2 ꢁ CH , C -11 + C -12); [27.2 (CH ), 24.3 (CH ), 23.3 (CH ), 0.80 (s, 6H, H -11 + H -12). C-NMR/DEPT-135 (CDCl , 101 MHz),

3

c

2

c

3

2.4 (CH ), 25.1 (CH ), 24.9 (CH ), C -4 + C -3 + C -3]. ESI-MS rotamers present, d ppm: [175.3 (Cq), 171.0 (Cq), 169.7 (Cq),

2

E

P

+

m/z: [M + H] calcd for [C30

H

49

N

4

O

7

] 577.36, found 577.35.

168.7 (Cq), C

E

-1 + C

); [60.9, 53.2, 52.0, 51.9 (2 ꢁ CH + CH

]; 51.9 (CH, C -5); 53.8 (Cq, C -1); [50.6 (CH

-2 + C ), C -2 + C -4 +

-5]; [46.4, 42.7, 39.9 (6 ꢁ CH

-8 + C -9 + C -3 + C -7); [30.4 (CH ),

-10]; 32.4 (2 ꢁ Cq, C

EtOAc. Aspect: light brown solid. Yield: 69% (0.4675 g). M.p.: 30.2 (CH ), C -4]; 30.2 (2 ꢁ CH , C -11 + C -12); [29.2 (CH ), 28.8

E

-5 + C

P

-1 + C

G

-1]; 155.8 (Cq, OCONH);

), C -2 +

),

Synthesis of Boc-Gly-Pro-Glu(OMe)-aminoindane (8a). General 79.8 (Cq, C(CH

3

)

3

P

ꢀ

protocol B, starting from 0.1470 g of L-proline. The organic phase

was dried with anhydrous Na SO , filtered and concentrated. The 47.3 (CH

resulting residue was purified by chromatographic column using -6 + C

C

E

-2 + CO

2

CH

3

c

2

4

2

), C

G

P

2

c

C

c

2

P

3

c

2

16

1

4

8–50 1C. [a]_D= ꢀ13.1 ꢂ 0.1 (c1, CHCl ). Analytical data: H-NMR (CH ), C -4]; 28.4 (3 ꢁ CH , C(CH ) ); [27.2 (CH ), 26.9 (CH ),

ꢀ

+

3

2

E

3

3 3

2

(CD OD, 400 MHz) d ppm: 7.28–7.12 (m, 4H, H -4 + H -5 + H -6 + C -3]; [25.1 (CH ), 24.9 (CH ), C -3]. ESI-MS m/z: [M + H] calcd

3 a a a E 2 2 P

+

H

3

a

-7); 5.36 (t, J = 7.6 Hz, 1H, H

.88 (d, J = 17.1 Hz, 1H, H -2); 3.75 (d, J = 17.1 Hz, 1H, H

CH ); 3.63–3.49 (m, 2H, H -5); 3.05 (ddd, J = 15.8, 8.7, protocol C, starting from 0.4582 g of 7a. Ia was purified by

.7 Hz, 1H, H -3); 2.87 (m, 1H, H -3); 2.56–1.77 (m, 10H, H -2 + chromatographic column using CH Cl /MeOH (9 : 1) as eluent.

-3 + H -4 + H -3 + H -4); 1.43 (s, 9H, C(CH ). C-NMR/DEPT- Aspect: white solid. Yield: 97% (0.4562 g). M.p.: 47–48 1C. [a]

35 (CD OD, 101 MHz) d ppm: [175.2 (Cq), 174.6 (Cq), 173.2 (Cq), ꢀ88.8 ꢂ 0.6 (c1, CH OH). Analytical data: H-NMR (CD OD,

a

-1); 4.43–4.29 (m, 2H, H

E

-2 + H

P

-2); for [C30

49 4 7

H N O ] 577.36, found 577.32.

G

-2); 3.67

Synthesis of H-Gly-Pro-Glu(aminoindane)-OMe (Ia). General

(s, 3H, CO

2

3

P

3

H

P

a

2

13

16

P

E

3 3

)

D

=

1

3

70.6 (Cq), C -1 + C -5 + C -1 + C -1]; 158.2 (Cq, CONH); [144.5 400 MHz) d ppm: 7.36–7.14 (m, 4H, H -4 + H -5 + H -6 + H -7);

E

P

G

a

(

Cq), 144.4 (Cq), C -3a + C -7a]; [128.9 (CH), 127.57 (CH), 125.7 5.37 (t, J = 7.6 Hz, 1H, H -1); 4.55–4.39 (m, 2H, H -2 + H -2); 3.74

a

E

P

(

[

CH), 125.2 (CH), C

62.0, 55.9, 54.4, 52.2, (3 ꢁ CH + CH

CO CH ); [47.8 (CH ), 43.9 (CH ), C

), 31.3 (CH ), 31.1 (CH ), 30.5 (CH

-4]; 28.7 (3 ꢁ CH , C(CH ); 27.7 (CH , C

a

-4 + C

a

-5 + C

a

-6 + C

a

-7]; 80.7 (Cq, C(CH

3

)

3

); (s, 3H, CO

2

CH

3

); 3.64–3.33 (m, 4H, 2H

P

-5 + 2H

G

-2); 2.99–2.77

ꢀ

3

), C

a

-1 + C

E P

-2 + C -2 + (m, 2H, H

a

-3); 2.57–1.75 (m, 10H, H

-4). C-NMR/DEPT-135 (CD OD, 101 MHz) d ppm: [174.6

-4 + (Cq), 174.6 (Cq), 174.2 (Cq), 173.5 (Cq), C -1 + C -5 + C -1 +

-3). -1]; [144.5 (Cq), 144.2 (Cq), C -3a + C -7a]; [128.9 (CH), 127.6

a P P E

-2 + H -3 + H -4 + H -3 +

1

3

2

3

2

P

-5 + C

G

-2]; 38.9 (CH, C

), C -2 + C -3 + C

-3); 25.9 (CH , C

P

-2);

H

E

3

[34.0 (CH

2

a

P

E

P

C

E

3

)

3

2

E

2

P

C

G

a

ꢀ

+

ESI-MS m/z: [M + H] calcd for [C H N O ] 531.28, found (CH), 125.7 (CH), 125.2 (CH), C -4 + C -5 + C -6 + C -7]; [61.5,

27

39

4

7

a

5

31.30.

55.8, 52.9, 52.8, (3 ꢁ CH + CH ), C -1 + C -2 + C -2 + CO CH ];

3

a

E

P

2

3

Synthesis of Boc-Gly-Pro-Glu(OMe)-amantadine (8b). General [47.5 (CH ), 43.3 (CH ), C -5 + C -2]; 33.0 (CH , C -2 + C -3);

2

P

G

2

a

protocol B, starting from 0.1691 g of L-proline. The resulting 31.0 (CH

2

, C

-3). ESI-MS m/z: [M] calcd for [C22

found 431.17.

Synthesis of H-Gly-Pro-Glu(amantadine)-OMe (Ib). General

00 MHz) d ppm: 7.60 (d, J = 7.6 Hz, 1H, CONH); 6.18 (br s, 1H, protocol C, starting from 0.4497 g of 7b. Aspect: yellow solid.

P

-4); 30.5 (CH

2

+

, C

E

-4); 28.6 (CH

2

, C

E

-3); 25.7

431.23,

+

residue was purified by chromatographic column using EtOAc. (CH

2

,C

P

H

31

N

4

O

5

]

16

Aspect: white solid. Yield: 65% (0.5239 g). M.p.: 56–58 1C. [a]

D

=

1

ꢀ

3 3

29.9 ꢂ 0.1 (c1, CHCl ). Analytical data: H-NMR (CDCl ,

4

1

6

CONH); 5.40 (br s, 1H, CONH); 4.45 (dd, J = 7.9 Hz, 1H, H -2); 4.28 Yield: 98% (0.4519 g). M.p.: 54–57 1C. [a] = +32.2 ꢂ 0.4 (c1,

E

D

1

(

td, J = 7.6, 4.4 Hz, 1H, H -2); 4.14–3.81 (m, 2H, H -2); 3.70–3.55 CH OH). Analytical data: H-NMR (CD OD, 400 MHz), rotamers

P G 3 3

(

m, 4H, CO CH + H -5); 3.48–3.37 (m, 1H, H -5); 2.53–2.34 present (80 : 20), d ppm: [4.50 (dd, J = 8.3, 3.4 Hz, major), 4.46

2 3 P P

(

H

(

m, 2H, H

-3); 1.65 (s, 6H, H

CDCl , 101 MHz) d ppm: [175.5 (Cq), 170.9 (Cq), 169.6 (Cq), 3H, CO

E

-4); 2.18–1.90 (m, 15H, H

E

-3 + H

P

1

-3 + H

P

-4 + H

). C-NMR/DEPT-135 3.89 (d, J = 3.2 Hz, 2H, H

CH ]; 3.67–3.49 (m, 2H, H

b

-2 + (dd, J = 8.7, 2.9 Hz, minor), 1H, H

-2); [3.73 (s, minor), 3.72 (s, major),

-5); 2.27–2.21 (m, 2H, H -4);

E P

-2]; 4.41–4.33 (m, 1H, H -2);

3

b

-4); 1.42 (s, 9H, C(CH

3

)

3

G

3

2

3

P

E

This journal is ©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2020

New J. Chem., 2020, 44, 21049--21063 | 21059

View Article Online

Paper

.19–1.82 (m, 15H, H

NJC

2

E

-3 + H

-4). C-NMR/DEPT-135 (CD

present, d ppm: [174.3 (Cq), 173.9 (Cq), 173.5 (Cq), 166.0 (Cq), 54.6, 54.4, 52.4, 52.3, (2 ꢁ CH + CH ), C -2 + C -2 + CO CH ];

P

-3 + H

P

-4 + H

b

-2 + H

b

-3); 1.71 101 MHz), rotamers present, d ppm: [175.2 (Cq), 173.9 (Cq),

1

3

(s, 6H, H

b

3 E E P G

OD, 101 MHz), rotamers 172.2 (Cq), 166.2 (Cq), C -1 + C -5 + C -1 + C -1]; [61.6, 61.2,

3

P

E

2

3

C -1 + C -5 + C -1 + C -1]; [61.4, 61.2, 53.8, 53.4, 52.8, (2 ꢁ CH + 53.1 (Cq, C -1); [47.7 (CH ), 42.3 (CH ), C -2 + C -5]; [41.5, 37.4,

E

P

G

b

2

G

P

CH ), C -2 + C -2 + CO CH ]; 52.8 (Cq, C -1); [48.5 (CH ), (6 ꢁ CH ), C -2 + C -4]; [31.3 (CH ), 31.2 (CH ), C -4]; 30.9

3

P

E

2

3

b

2

b

2

P

4

C

3

(

7.7 (CH

-2 + C

0.7 (CH

CH ), C

found 449.31.

2

), C

G

-2 + C

P

-5]; [42.3, 42.3, 41.4, 37.5, (6 ꢁ CH

), 33.9 (CH ), C

-4]; 30.9 (3 ꢁ CH, C

-4); [28.8 (CH ), 27.9 (CH ), C -3]; [25.5 (CH

-3]. ESI-MS m/z: [M] Calcd for [C23

2

), (3 ꢁ CH, C

b

-3); 30.8 (CH

), 23.4 (CH ), C

] 449.28, found 449.33.

2

, C

E

2 2 E

-4); [28.7 (CH ), 28.3 (CH ), C -3];

P

-3]. ESI-MS m/z: [M] Calcd for

+

b

-4]; [34.0 (CH

, C

2

P

b

-3); [25.7 (CH

), 23.4 [C23

2

+

2

E

2

E

2

H

37

N

4

O

5

+

2

P

H

37

N

4

O

5

] 449.28,

Synthesis of H-Gly-Pro-Glu(OMe)-memantine (IIc). General

protocol C, starting from 0.2181 g of 8c. Aspect: brown solid.

1

D

9

Synthesis of H-Gly-Pro-Glu(memantine)-OMe (Ic). General Yield: 93% (0.2078 g). M.p.: 130–135 1C. [a] = ꢀ26.9 ꢂ 0.2 (c1,

1

protocol C, starting from 0.4011 g of 7c. Aspect: white solid with CH OH). Analytical data: H-NMR (CD OD, 400 MHz), rotamers

3

1

D

9

low melting point. Yield: 95% (0.3903 g). [a] = ꢀ36.1 ꢂ 0.2 present (80 : 20), d ppm: 4.51–4.43 (m, 1H, H -2); 4.32–4.24

E

1

(

c1, CH

rotamers present (80 : 20), d ppm: 4.58–4.30 (m, 2H, H

-2); 3.90 (d, J = 7.5 Hz, 2H, H -2); [3.73 (s, minor), 3.72 12H), 1.69–1.60 (m, 3H), 1.36 (dd, J = 30.6, 12.3 Hz, 4H), 1.16 (s,

s, major), 3H, CO CH ]; 3.64–3.45 (m, 2H, H -5); [2.52–1.73 2H), H -3 + H -4 + H -3 + H -4 + H -2 + H -4 + H -5 + H -6 + H -8 +

m, 11H), 1.64 (q, J = 11.9 Hz, 4H), 1.36 (dd, J = 29.8, 11.4 Hz, -9 + H -10]; 0.85 (s, 6H, H -11 + H -12). C-NMR/DEPT-135

H), 1.16 (s, 2H), H -3 + H -4 + H -3 + H -4 + H -2 + H -4 + (CD OD, 101 MHz), rotamers present, d ppm: [175.2 (Cq), 173.9

3

OH). Analytical data: H-NMR (CD

3

OD, 400 MHz), (m, 2H, H

P

-2); 3.87 (d, 2H, H

G

-2); [3.68 (s, minor), 3.67 (s,

]; 3.63–3.44 (m, 2H, H -5); [2.51–1.80 (m,

3 P

E

-2 + major), 3H, CO

2

CH

H

(

P

G

2

3

P

E

c

1

3

H

c

4

P

E

c

3

H -5 + H -6 + H -8 + H -9 + H -10]; 0.85 (s, 6H, H -11 + H -12). (Cq), 172.3 (Cq), 166.3 (Cq), C -1 + C -5 + C -1 + C -1]; [61.7, 54.4,

c

E

P

G

1

3

C-NMR/DEPT-135 (CD OD, 101 MHz), rotamers present, d 52.3, (2 ꢁ CH + CH ), C -2 + C -2 + CO CH ]; 54.7 (Cq,

3

P

E

2

3

ppm: [174.0 (Cq), 173.5 (Cq), C

Cq, C

CO CH

), 34.1 (CH

-10]; 33.2 (2 ꢁ Cq, C

E

-1 + C

E

-5 + C

), C

-5]; [43.8 (CH

-6 + C -8 + C

P

-1 + C

-2 + C

), 40.8 (2 ꢁ Cq, C

-9 +

),

G

-1]; 54.5

C

c

-1); [51.7 (CH

2

), 48.2 (CH

2

), C

G

-2 + C

P

-5]; [47.7, 43.7, 41.5,

-9 + C -10]; 33.2

), C

-4]; [30.7 (CH

-3]; [25.6 (CH ), 23.4 (CH

(

c

-1); [53.5, 52.9, 52.8, (2 ꢁ CH + CH

3

P

E

-2 + 40.7 (6 ꢁ CH

2

), C -2 + C -4 + C

c

-6 + C

c

-8 + C

c

2

3

]; [51.7 (CH

2

), 48.3 (CH

2

), C

G

-2 + C

P

2

c

-3 + C

c

-7); [31.5, 30.7, (CH + 2 ꢁ CH

3

c

-5 + C

c

-11 +

),

-3].

(

C

CH

2

), 33.9 (CH

2

), C

c

-2 + C

c

-4 + C

c

C

c

-12]; [31.3 (CH

-4]; [28.7 (CH ), 28.4 (CH

2

), 31.1 (CH

2

), C

P

2

), 31.1 (CH

2

c

-3 + C

c

-7); [31.6, 30.7, (CH + 2 ꢁ CH

3

C

E

2

+

2

), C

E

2

), C

P

+

C -5 + C -11 + C -12]; 30.7 (CH , C -4); 28.8 (CH , C -4); 27.9 ESI-MS m/z: [M] calcd for [C H N O ] 477.31, found 477.34.

c

2

P

2

E

25 41 4 5

+

(

[

CH , C -3); 23.5 (CH , C -3). ESI-MS m/z: [M] calcd for

Synthesis of Boc-Pro-Glu(OMe)-amantadine (9b). To

solution of Boc-Pro-OH (0.1091 g, 0.5069 mmol) in anhydrous

Synthesis of H-Gly-Pro-Glu(OMe)-aminoindane (IIa). General CH Cl (25 mL) prepared in a round-bottom flask was subse-

protocol C, starting from 0.4352 g of 8a. IIa was purified by quentially added Et N (0.21 mL, 1.5 mmol), TBTU (0.1790 g,

chromatographic column using CH Cl /MeOH (9 : 1) as eluent. 0.5575 mmol) and 4b (0.2484 g, 0.6083 mmol). The resulting

Aspect: brown solid. Yield: 98% (0.4377 g). M.p.: 52–54 1C. [a]

a

2

E

2

P

+

C H N O ] 477.31, found 477.31.

25 41 4 5

2

3

2

1

D

7

=

solution was stirred for 2 h at RT under an inert atmosphere

OD, (Ar). After the reaction, was followed the same purification

00 MHz), rotamers present, d ppm: 7.30–7.08 (m, 4H, H -4 + method described in the general protocol B, using EtOAc for

1

ꢀ

11.7 ꢂ 0.8 (c1, CH

3 3

OH). Analytical data: H-NMR (CD

4

a

H -5 + H -6 + H -7); 5.38 (t, J = 7.5 Hz, 1H, H -1); 4.51–4.27 (m, 2H, the chromatographic column. Aspect: yellow oil. Yield: 96%

a

2

H -2 + H -2); 3.67 (s, 3H, CO CH ); 3.64–3.35 (m, 4H, 2H -2 + (0.2394 g). [a] = ꢀ71.4 ꢂ 0.3 (c1, CHCl ). Analytical data:

E

P

2

3

G

D

3

1

2

H

P

-5); 3.13–2.73 (m, 2H, H

-4 + H

a

-3); 2.60–1.72 (m, 10H, H

-4). C-NMR/DEPT-135 (CD

a

-2 + H

P

-3 +

H-NMR (CDCl

3

, 400 MHz), rotamers present (50 : 50) d ppm:

13

H

P

E

-3 + H

E

3

OD, 101 MHz), [6.26 (s), 6.04 (s), 1H, CONH); 4.38–4.10 (m, 2H, H

-1 + 3.66 (s, 3H, CO CH ); 3.55–3.30 (m, 2H, H -5); 2.52–2.31 (m, 2H,

-7a]; -4); 2.20–1.79 (m, 15H, H -3 + H -3 + H -4 + H -2 + H -3); 1.65

-5 + (s, 6H, H -4); 1.44 (s, 9H, C(CH ). C-NMR/DEPT-135 (CDCl

C -6 + C -7]; [61.9, 55.8, 54.3, 52.3, (3 ꢁ CH + CH ), C -1 + C -2 + 101 MHz), rotamers present, d ppm: [174.5 (Cq), 172.4 (Cq),

E P

-2 + H -2);

rotamers present, d ppm: [175.2, 174.4, 173.2, (4 ꢁ Cq), C

-5 + C -1 + C -1]; [144.6 (Cq), 144.3 (Cq), C -3a + C

128.9 (CH), 127.6 (CH), 125.7 (CH), 125.1 (CH), C -4 + C

E

2

3

P

C

[

E

P

G

a

H

E

P

b

1

3

a

b

3

)

3

,

a

3

a

E

C -2 + CO CH ]; [47.6 (CH ), 42.6 (CH ), C -5 + C -2]; 31.2 (CH , 169.8 (Cq), C -1 + C -5 + C -1]; 80.6 (Cq, C(CH ) ); [60.8, 53.3,

P

2

3

2

P

G

2

E

P

ꢀ

3 3

C -4); 31.1 (CH , C -2 + C -3); 30.7 (CH , C -4); 27.9 (CH , C -3); 51.9, (2 ꢁ CH + CH ), C -2 + C -2 + CO CH ]; 52.3 (Cq, C -1);

P

2

a

2

E

2

+

E

3

P

E

2

3

b

+

2

5.7 (CH

found 431.19.

Synthesis of H-Gly-Pro-Glu(OMe)-amantadine (IIb). General C(CH

2

, C

P

-3). ESI-MS m/z: [M] calcd for [C22

H

31

N

4

O

5

] 431.23, [47.2 (CH

-5 + C

); [27.2 (CH

2

), 41.6 (CH

2

), 36.5 (CH

-4]; 29.5 (3 ꢁ CH, C

), 24.7 (CH ), C -3 + C

] 492.31, found 492.29.

Synthesis of H-Pro-Glu(OMe)-amantadine (10b). General

c1, CH OH). Analytical data: H-NMR (CD OD, 400 MHz), protocol C, starting from 0.1318 g of 9b. Aspect: colourless oil.

2

), 30.3 (CH

2

), 30.3 (CH ),

C

P

b

-2 + C

b

-4 + C

P

b

-3); 28.5 (3 ꢁ CH

3

,

ꢀ

3

)

3

+

2

E

P

-3]. ESI-MS m/z:

+

42 3 6

protocol C, starting from 0.4230 g of 8b. Aspect: white solid. [M + H] calcd for [C26H N O

1

6

Yield: 98% (0.4251 g). M.p.: 67–69 1C. [a]

D

= ꢀ31.2 ꢂ 0.2

1

(

3

2

3

rotamers present (85 : 15), d ppm: 4.51–4.43 (m, 1H, H -2); Yield: 97% (0.1311 g). [a] = ꢀ9.9 ꢂ 0.2 (c1, CH OH). Analytical

E

D

3

1

4

.31–4.23 (m, 1H, H -2); 3.89 (s, 2H, H -2); [3.68 (s, minor), data: H-NMR (CD OD, 400 MHz), rotamers present (70:30),

P

G

3

.67 (s, major), 3H, CO

2

CH

3

]; 3.63–3.49 (m, 2H, H

P

-5); 2.52–2.34 d ppm: 4.22–4.13 (m, 2H, H

-4 + H -2 + minor), 3H, CO CH ]; 3.33–3.20 (m, 2H, H

C-NMR/DEPT-135 (CD OD, -4 + H -3); 1.96–1.86 (m, 13H, H -3 + H

E

-2 + H

P

-2); [3.54 (s, major), 3.53 (s,

-5); 2.40–2.20 (m, 4H,

-4 + H -2 + H -3); 1.58

(m, 2H, H

E

-4); 2.11–1.85 (m, 15H, H

E

-3 + H

P

-3 + H

P

b

2

3

P

1

3

H

b

-3); 1.72 (s, 6H,

H

b

-4).

3

H

E

P

b

2

1060 | New J. Chem., 2020, 44, 21049--21063

View Article Online

NJC

Paper

1

3

(

s, 6H, H

present, d ppm: [174.9 (Cq), 171.8 (Cq), 169.6 (Cq), C

C -1]; [60.9, 54.7, 52.3 (2 ꢁ CH + CH3), C -2 + C -2 + CO CH ]; 53.1 H -4 + H -2 + H -4 + H -5 + H -6 + H -8 + H -9 + H -10]; 0.81 (s, 6H,

b

-4). C-NMR/DEPT-135 (CD

3

OD, 101 MHz), rotamers (s, minor), 2.48 (s, major), 6H, (CH

3

)

2

N]; [2.25–1.74 (m, 11H),

E

-1 + C -5 + 1.60 (q, J = 11.8 Hz, 4H), 1.39–1.07 (m, 6H), H

E

P

-3 + H -4 + H -3 +

P

E

P

E

2

3

E

c

13

(

C

3

Cq, C -1); 48.4 (CH , C -5); [42.3, 37.4, (6 ꢁ CH ), C -2 + C -4]; 31.0 H -11 + H -12). C-NMR/DEPT-135 (CDCl , 101 MHz), rotamers

b

2

P

2

b

c

3

CH , C -4); 30.9 (3 ꢁ CH, C -3); [28.7 (CH ), 24.9 (CH ), C -4, C -3, present, d ppm: [172.5 (Cq), 172.3 (Cq), 171.9 (Cq), 171.8 (Cq),

2

P

b

2

E

+

P

-3]. ESI-MS m/z: [M] calcd for [C21

92.30.

Synthesis of (N,N-dimethyl-Gly)-Pro-Glu(aminoindane)-OMe

IVa). General protocol D, starting from 0.3343 g of Ia. Purifica-

tion: chromatographic column using CH Cl

/MeOH (4 : 1) as (CH + 2 ꢁ CH

eluent. Aspect: yellow solid. Yield: 72% (0.2027 g). M.p.: 26.8 (CH ), C -4]; 25.0 (CH , C -3); 22.6 (CH , C -3). ESI-MS m/z:

H

34

3 4

N O ]

392.25, found

C

E

-1 + C

-2 + CO

-2 + C

-5]; [42.8, 39.9, 33.7, 33.4, 32.1 (6 ꢁ CH

-6 + C -8 + C -9 + C -3 + C

-10]; 32.4 (2 ꢁ Cq, C

), C -5 + C -11 + C -12]; 28.9 (CH , C

E

-5 + C

P

-1 + C

G

-1]; [60.7, 52.7, 52.5, (2 ꢁ CH + CH

-1); [50.7 (CH ), 47.5 (CH ), 47.2 (CH

), C -2 + C -4 +

-7); [30.2, 30.2,

-4); [28.2 (CH ),

3

), C

P

-2 +

2

),

2

CH ]; 53.6 (Cq, C

3

c

2

C

G

P

2

c

(

C

c

2

3

c

2

P

2

E

2

E

2

P

2

+

3

9–41 1C. [a]_D= +68.6 ꢂ 0.1 (c1, CH OH). Analytical data: [M + H] calcd for [C H N O ] 505.34, found 505.31.

3

27 45 4 5

1

H-NMR (CD OD, 400 MHz), rotamers present (80 : 20),

Synthesis of (N,N-dimethyl-Gly)-Pro-Glu(OMe)-aminoindane,

-7); 5.38 (t, Va. General protocol D, starting from 0.3221 g of IIa. Purification:

-2 + H -2); [3.75 chromatographic column using CH Cl /MeOH (4 : 1) as eluent.

]; 3.63–3.33 (m, 4H, 2H -2 + Aspect: yellow oil. Yield: 70% (0.1899 g). [a]

-3); 2.49–1.89 (m, 16H, (CH

3

d ppm: 7.28–7.14 (m, 4H, H

J = 7.5 Hz, 1H, H -1); 4.51–4.39 (m, 2H, H

s, minor), 3.73 (s, major), 3H, CO CH

-5); 3.05–2.79 (m, 2H, H

-2 + H -3 + H -4 + H -3 + H

01 MHz), rotamers present, d ppm: [174.6 (Cq), 174.5 (Cq), H -7); 5.38 (t, J = 7.6 Hz, 1H, H -1); 4.51–4.30 (m, 2H, H -2 + H -2);

a a a a

-4 + H -5 + H -6 + H

a

E

P

2

(

2

H

1

2

3

G

D

= ꢀ12.2 ꢂ 0.1 (c1,

OD, 400 MHz), rotamers

OD, present (80 : 20), d ppm: 7.29–7.09 (m, 4H, H -4 + H -5 + H -6 +

1

H

P

a

3 2 3 3

) -N + CH OH). Analytical data: H-NMR (CD

1

3

a

P

E

-4). C-NMR/DEPT-135 (CD

3

a

E

P

1

74.4 (Cq), 173.5 (Cq), C -1 + C -5 + C -1+ C -1]; [144.5 (Cq), [3.68 (minor), 3.67 (s, major), 3H, CO CH ]; 3.59–3.34 (m, 2H,

E E P G 2 3

1

44.4 (Cq), C -3a + C -7a]; [128.8 (CH), 127.7 (CH), 125.6 (CH), H -2); 3.10–2.79 (m, 3H, H -3); [2.71–1.80 (m, 17H), 1.35–1.24

a

G

a

1

25.2 (CH), C

a

-4 + C

-1 + C

), 45.4 (CH

), 34.3 (CH ), C -5]; [33.1 (CH

-2 + C -3); 30.5 (CH , C -4); [28.7 (CH

C -3]; [25.8 (CH ), 23.3 (CH ), C -3]. ESI-MS m/z: [M + H] calcd CH ), C -1 + C -2 + C -2 + CO CH ]; [48.3 (CH ), 48.0 (CH ), C -2];

a

-5 + C

a

-6 + C

-2 + CO

), 45.3 (CH

a

-7]; [61.5, 55.8, 53.1, 52.8, (3 ꢁ (m, 1H), H

P

-5 + H

C-NMR/DEPT-135 (CD

-N]; [175.2, 173.2, (4 ꢁ Cq), C

-4]; 31.0 (Cq), C -3a + C -7a]; [128.9 (CH), 127.6 (CH), 125.7 (CH), 125.1

), 27.8 (CH ), (CH), C -4 + C -5 + C -6 + C

-7]; [61.8, 55.8, 54.3, 52.3, (3 ꢁ CH +

a

-2 + (CH

OD, 101 MHz), rotamers present, d ppm:

-1 + C -5 + C -1 + C -1]; [144.5 (Cq), 144.3

3 2 P P E E

) -N + H -3 + H -4 + H -3 + H -4].

13

CH + CH

3

), C

a

E

-2 + C

P

2

CH

3

]; [48.3 (CH

), 45.3 (CH

), 32.9 (CH

2

), 48.0 (CH

), (CH

), C

2

),

3

C

G

-2]; [45.5 (CH

34.4 (CH

CH , C

3

)

2

E

P

G

[

(

2

P

2

P

a

2

a

2

E

2

a

+

E

2

P

3

a

E

P

2

3

2

G

+

for [C H N O ] 459.26, found 459.31.

[45.5 (CH ), 45.5 (CH ), 45.4 (CH ), (CH ) -N]; [34.2 (CH ), 33.9

3 3 3 3 2 2

2

4

35

4

5

Synthesis of (N,N-dimethyl-Gly)-Pro-Glu(amantadine)-OMe, (CH ), C -5]; [31.3 (CH ), 31.2 (CH ), C -4]; 31.1 (CH , C -2 + C -3);

2

P

2

P

2

a

IVb. General protocol D, starting from 0.3323 g of Ib. Purifica- 30.6 (CH

tion: chromatographic column using CH Cl /MeOH (4 : 1) as ESI-MS m/z: [M + H] calcd for [C24

eluent. Aspect: white solid. Yield: 72% (0.2027 g). M.p.:

2

, C

E

-4); 28.1 (CH

2

, C

E

-3); [26.0 (CH

H N O ] 459.26, found 459.31.

35 4 5

2

), 25.8 (CH

2

), C

P

-3].

+

2

Synthesis of (N,N-dimethyl-Gly)-Pro-Glu(OMe)-amantadine

2

0

7

9–81 1C. [a]

D

= ꢀ28.8 ꢂ 0.2 (c1, CH

3

OH). Analytical data: (Vb). General protocol D, starting from 0.3208 g of IIb.

1

H-NMR (CD

3

OD, 400 MHz), rotamers present (80 : 20), d ppm: Purification: chromatographic column using CH

2 2

Cl /MeOH

4

.52–4.44 (m, 1H, H -2); 4.39 (dd, J = 9.4, 4.8 Hz, 1H, H -2); (4 : 1) as eluent. Aspect: white solid. Yield: 70% (0.1903 g).

E

P

2

0

.02–3.93 (m, 2H, H -2); [3.74 (s, minor), 3.72 (s, major), 3H, M.p.: 67–69 1C. [a] = +23.9 ꢂ 0.2 (c1, CH OH). Analytical data:

G

D

3

1

CO CH ]; 3.65–3.50 (m, 2H, H -5); 2.81 (s, 6H, (CH ) N);

H-NMR (CD OD, 400 MHz), rotamers present (85 : 15), d ppm:

3

2

3

P

3 2

2

H

(

5

.28–2.20 (m, 2H, H

-4 + H -2 + H

CD OD, 101 MHz), rotamers present, d ppm: [174.3 (Cq), 173.9 (s, major), 3H, CO

Cq), 173.5 (Cq), 165.9 (Cq), C -1 + C -5 + C -1 + C -1]; [61.5, (CH N); 2.50–2.37 (m, 2H, H

3.8, 53.3 (2 ꢁ CH + CH ), C -2 + C -2 + CO CH ]; 52.8 (Cq, -3 + H -4 + H -2 + H -3); 1.72 (s, 6H, H

E

-4); 2.14–1.87 (m, 15H, H

E

-3 + H

P

-3 + 4.46 (dd, J = 8.6, 4.1 Hz, 1H, H

-2); 3.99 (d, J = 3.8 Hz, 2H, H

CH ]; 3.63–3.48 (m, 2H, H

-4); 2.27–1.86 (m, 15H, H

E

-2); 4.29 (dd, J = 8.9, 5.1 Hz, 1H,

-2); [3.69 (s, minor), 3.67

-5); 2.83 (s, 6H,

-3 +

-4). C-NMR/DEPT-

1

3

P

b

-3); 1.71 (s, 6H, H

b

-4). C-NMR/DEPT-135

H

P

G

3

2

3

P

E

P

G

3

)

2

E

1

3

P

E

2

3

H

P

b

C -1); [59.9 (CH ), 47.9 (CH ), C -2 + C -5]; 45.1 (2 ꢁ CH , 135 (CD OD, 101 MHz), rotamers present, d ppm: [173.8 (Cq),

b

2

G

P

3

(

CH ) N); [42.3, 37.5, (6 ꢁ CH ), C -2 + C -4]; [34.0 (CH ), 33.1 172.6 (Cq), 170.8 (Cq), 164.5 (Cq), C -1 + C -5 + C -1 + C -1];

3

2

b

2

E

P

G

(

2

CH ), C -4]; 30.9 (3 ꢁ CH, C -3); 30.7 (CH , C -4); [28.8 (CH ), [60.3, 59.9, 53.2, 52.9, 51.0, 50.8, (2 ꢁ CH + CH ), C -2 + C -2 +

2

P

b

2

E

2

3

P

E

7.7 (CH

2

), C

E

-3]; [25.6 (CH

2

), 23.4 (CH

2

), C

P

-3]. ESI-MS m/z: CO

2

CH

-2 + C

3

]; 51.7 (Cq, C

b

-1); [58.8 (CH

-5]; 43.6 (2 ꢁ CH , (CH

-4]; [29.9 (CH ), 29.7 (CH

-3); [27.4 (CH ), 26.8 (CH

-3]. ESI-MS m/z: [M + H] calcd for [C25

477.31, found 477.27.

Synthesis of (N,N-dimethyl-Gly)-Pro-Glu(OMe)-memantine

00 MHz), rotamers present (75 : 25), d ppm: [8.62 (br s, minor), (Vc). General protocol D, starting from 0.1078 g of IIc. Purification:

.60 (br s, major), 1H, CONH]; [6.05 (br s, major), 5.90 (br s, chromatographic column using CH Cl /MeOH (4 : 1) as eluent.

-2 + H -2); [3.75–3.63 Aspect: white solid. Yield: 60% (0.0553 g). M.p.: 60–63 1C.

+ H -2 + H

2

), 58.4 (CH

2

), 46.4 (CH

2

),

+

[M + H] calcd for [C25

H

41

N

4

O

5

] 477.31, found 477.26.

C

G

P

b

3

)

2

N); [40.9, 36.0, (6 ꢁ CH

), C -4]; 29.3 (CH , C

), C -3]; [24.4 (CH

2

),

Synthesis of (N,N-dimethyl-Gly)-Pro-Glu(memantine)-OMe

b

2

P

2

E

-4);

),

(

IVc). General protocol D, starting from 0.2914 g of Ic. Purification: 29.5 (3 ꢁ CH, C

Cl /MeOH (4 : 1) as eluent. 21.9 (CH ), C

Aspect: white solid. Yield: 60% (0.1494 g). M.p.: 73–75 1C. [a]

b

2

E

2

+

chromatographic column using CH

2

P

41 4 5

H N O ]

1

9

=

D

1

ꢀ

57.1 ꢂ 0.2 (c1, CH OH). Analytical data: H-NMR (CDCl₃,

3

4

7

2

minor), 1H, CONH]; 4.45 (d, J = 5.8 Hz, 2H, H

m, 4H), 3.60–3.34 (m, 3H), CO CH

E

P

1

9

1

(

2

3

G

P

-5]; [2.71 [a]

D

= ꢀ42.6 ꢂ 0.3 (c1, CH

3

OH). Analytical data: H-NMR

New J. Chem., 2020, 44, 21049--21063 | 21061

View Article Online

Paper

NJC

(

CDCl

3

, 400 MHz), rotamers present (70 : 30), d ppm: 7.49

8 P. B. Watkins, H. J. Zimmerman, M. J. Knapp, S. I. Gracon

and K. W. Lewis, JAMA, 1994, 271, 992–998.

d, J = 7.8 Hz, 1H, CONH); 6.29 (s, 1H, CONH); 4.57–4.21 (m,

2

3

H, H -2 + H -2); [3.65 (s, minor), 3.64 (s, major), 3H, CO CH ];

9 H. Tang, L. Z. Zhao, H. T. Zhao, S. L. Huang, S. M. Zhong,

J. K. Qin, Z. F. Chen, Z. S. Huang and H. Liang, Eur. J. Med.

Chem., 2011, 46, 4970–4979.

E

P

2

3

.58–3.35 (m, 4H, H -2 + H -5); 2.56 (s, 6H, (CH ) N); [2.14–1.77

G

P

3 2

(m, 10H), 1.61 (s, 4H), 1.38–1.06 (m, 7H), H -3 + H -4 + H -3 +

P P E

H

E

-4 + H

-11 + H

rotamers present, d ppm: [174.5 (Cq), 171.6 (Cq), 170.1 (Cq), 11 D. C. G o´ recki, M. Ber ˛e sewicz and B. Zabłocka, Neurochem.

71.8 (Cq), C -1 + C -5 + C -1 + C

Int., 2007, 51, 451–458.

CH ), C -2 + C -2 + CO CH

), 47.3 12 J. Guan, P. Harris, M. Brimble, Y. Lei, J. Lu, Y. Yang and

A. J. Gunn, Expert Opin. Ther. Targets, 2015, 19, 785–793.

c

-2 + H

c

-4 + H

c

-5 + H

c

-6 + H

c

-8 + H

c

-9 + H

, 101 MHz),

c

-10] 0.81 (s, 10 A. H. Dyer, C. Vahdatpour, A. Sanfeliu and D. Tropea,

Neuroscience, 2016, 325, 89–99.

3

1

3

6H, H

c

-12). C-NMR/DEPT-135 (CDCl

1

E

P

G

-1]; [60.8, 53.4, 51.9, (2 ꢁ CH +

3

P

E

2

3

c 2

]; 53.9 (Cq, C -1); [50.7 (CH

(

CH ), 47.1 (CH ), C -2 + C -5]; [42.7, 40.1, 39.9, (6 ꢁ CH ),

2

G

P

2

C -2 + C -4 + C -6 + C -8 + C -9 + C -10]; 32.4 (2 ꢁ Cq, C -3 + 13 S. A. Alonso De Diego, M. Guti ´e rrez-Rodr ´ı guez, M. J. P ´e rez de

c

C -7); [30.4 (CH ), 32.1 (CH ), C -4]; [30.2, (CH + 2 ꢁ CH ), C -5 +

Vega, R. Gonz ´a lez-Mu n˜ iz, R. Herranz, M. Mart ´ı n-Mart ´ı nez,

E. Cenarruzabeitia, D. Frechilla, J. Del R ´ı o, M. L. Jimeno and

M. T. Garc ´ı a-L o´ pez, Bioorg. Med. Chem. Lett., 2006, 16,

3396–3400.

c

2

P

3

c

C

c

-11 + C

c

-12]; 28.9 (CH

2

, C

E

-4); 27.4 (CH

2

, C

E

-3); [25.0 (CH

2

),

+

22.5 (CH

2

), C

P

-3]. ESI-MS m/z: [M + H] calcd for [C27

H

45

N

4

O

5

]

505.34, found 505.33.

1

4 X.-C. M. Lu, R.-W. Chen, C. Yao, H. Wei, X. Yang, Z. Liao, J. R.

Dave and F. C. Tortella, J. Neurotrauma, 2009, 26, 141–154.

5 J. Guan and P. D. Gluckman, Br. J. Pharmacol., 2009, 278,

Author contributions

85–90.

Conceived and designed the experiments: SCSR, IESD. Per-

formed the experiments: SCSR, ACVDS. Analysed the data:

SCSR, IESD, XGM and JERB. Wrote the paper: SCSR and IESD.

All authors have given approval to the final version of the

manuscript. The authors declare no competing financial

interest.

6 M. J. Bickerdike, G. B. Thomas, D. C. Batchelor, E. S.

Sirimanne, W. Leong, H. Lin, F. Sieg, J. Wen, M. A.

Brimble, P. W. Harris and P. D. Gluckman, J. Neurol. Sci.,

2009, 278, 85–90.

1

7 Neuren Pharmaceuticals, Annual Report, 2008.

8 M. A. Brimble, N. S. Trotter, P. W. R. Harris and F. Sieg,

Bioorg. Med. Chem., 2005, 13, 519–532.

1

9 M. Y. H. Lai, M. A. Brimble, D. J. Callis, P. W. R. Harris, M. S.

Levi and F. Sieg, Bioorg. Med. Chem., 2005, 13, 533–548.

Conflicts of interest

There are no conflicts to declare.

20 S. A. Alonso De Diego, M. Guti ´e rrez-Rodr ´ı guez, M. J. P ´e rez

de Vega, D. Casabona, C. Cativiela, R. Gonz ´a lez-Mu n˜ iz,

R. Herranz, E. Cenarruzabeitia, D. Frechilla, J. D. R ´ı o,

Acknowledgements

´

M. Luisa Jimeno and M. Teresa Garcıa-Lopez, Bioorg. Med.

Chem. Lett., 2006, 16, 1392–1396.

This research was funded by Fundaç ˜a o para a Ci ˆe ncia e

Tecnologia (FCT, Portugal), through grants UIDB/50006/2020

2

1 I. Cacciatore, L. Baldassarre, E. Fornasari, C. Cornacchia,

A. Di Stefano, P. Sozio, L. S. Cerasa, A. Fontana, S. Fulle,

E. S. Di Filippo, R. M. L. La Rovere and F. Pinnen, Chem-

MedChem, 2012, 7, 2021–2029.

(to LAQV-REQUIMTE Research Unit) and PTDC/BIA-MIB/29059/

2

017. SCSR thanks FCT for the PhD grant SFRH/BD/147463/

019. The authors thank Mariana Andrade for the NMR experi-

2

2 D. C. Batchelor, H. Lin, J. Y. Wen, C. Keven, P. L. V. Zijl,

B. H. Breier, P. D. Gluckman and G. B. Thomas, Anal.

Biochem., 2003, 323, 156–163.

3 I. Cacciatore, C. Cornacchia, L. Baldassarre, E. Fornasari,

A. Mollica, A. Stefanucci and F. Pinnen, Mini Rev. Med. Chem.,

2012, 12, 13–23.

ments at CEMUP.

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Article Doi

Bioinspired design for the assembly of Glypromate neuropeptide conjugates with active pharmaceutical ingredients

DOI: 10.1039/d0nj04851h

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Article abstract of DOI:10.1039/d0nj04851h

Full text of DOI:10.1039/d0nj04851h

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