Structure-activity relationship studies on the inhibition of the bacterial translation of novel Odilorhabdins analogues

B i o o r g an i c &M e d i c i n a l C h e m i s tr y x x x (x xxx )x xxx

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Bioorganic & Medicinal Chemistry

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

Structure-activity relationship studies on the inhibition of the bacterial

translation of novel Odilorhabdins analogues

Einars Loza^a, Matthieu Sarciaux^b, Martins Ikaunieks^a, Martins Katkevics^a, Tatyana Kukosha^a,

Nadezhda Truﬁlkina^a, Victoria Ryabova^a, Kirill Shubin^a, Lucile Pantel^b, Marine Serri^b,

Douglas L Huseby^c, Sha Cao^c, Kavita Yadav^c, Karin Hjort^c, Diarmaid Hughes^c, Maxime Gualtieri^b,

⁎

Edgars Suna^a,, Emilie Racine^b,

^aLatvian Institute of Organic Synthesis, Aizkraukles 21, LV-1006 Riga, Latvia

^bNosopharm, 110 allée Charles Babbage, Espace Innovation 2, 30000 Nîmes, France

^cDepartment of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Uppsala, Sweden

A R T I C L E I N F O

A B S T R A C T

Keywords:

A structure–activity relationship (SAR) study of NOSO-95179, a nonapeptide from the Odilorhabdin class of

antibacterials, was performed by systematic variations of amino acids in positions 2 and 5 of the peptide. A series

of non-proteinogenic amino acids was synthesized in high enantiomeric purity from Williams’ chiral dipheny-

loxazinone by highly diastereoselective alkylation or by aldol-type reaction. NOSO-95179 analogues for SAR

studies were prepared using solid-phase peptide synthesis. Inhibition of bacterial translation by each of the

synthesized Odilorhabdin analogues was measured using an in vitro test. For the most eﬃcient analogues, an-

tibacterial eﬃcacy was measured against two wild-type Enterobacteriaceae (Escherichia coli and Klebsiella pneu-

moniae) and against an eﬄux defective E. coli strain (ΔtolC) to evaluate the impact of eﬄux on the antibacterial

activity.

Odilorhabdins

Novel antibiotic class

SAR

Inhibition of bacterial translation

Novel amino acids

1. Introduction

mechanisms.^6,7Odilorhabdins (ODLs) have been discovered recently

and are a novel antibiotic class with a broad-spectrum activity against

Antimicrobial resistance is one of the serious threats to public health

the world is facing. Recently, the World Health Organization (WHO)

has published a list of antibiotic resistant pathogens for which drug

discovery and development of new treatment options are urgently

needed.¹As part of this list, carbapenem-resistant Enterobacteriaceae

(CRE) have been classiﬁed as critical threats as they are resistant to all

or nearly all of the antibacterials available. CRE have caused numerous

outbreaks of severe nosocomial infections and have become endemic in

several countries.^2,3Recent reports show that patients with CRE in-

fections are approximately 3 times more likely to die than patients with

infections that are susceptible to carbapenems.⁴In 2013, according to

the CDC's data, approximately 600 deaths and 9,300 healthcare-asso-

ciated infections were caused in the United States by the two most

common types of CRE, carbapenem-resistant Klebsiella species and Es-

cherichia coli.⁵

Gram-positive and Gram-negative pathogens, including critical patho-

gens as deﬁned by the WHO.^1,8,9,10ODLs are a family of peptides that

inhibit speciﬁcally the bacterial translation. Following a hit-to-lead

program to study the structure–activity relationships (SAR) of these

peptides, NOSO-95179 1 (Fig. 1) was identiﬁed as a lead compound

showing promising in vitro and in vivo antibacterial eﬃcacy against

Enterobacteriaceae (including carbapenem-resistant Enterobacteriaceae)

and low toxicity.¹¹NOSO-95179 1 is a 9-mer linear cationic peptide

containing six proteinogenic amino acids and three non-proteinogenic

amino acids: ((2S,3S)-α,γ-diamino β-hydroxy butyric acid (Dab(βOH))

in position 2, ^D-ornithine (D-Orn) in position 5 and Z-α,β-dehy-

droarginine (Dha) in position 9. Based on the MIC₉₀of NOSO-95179

against E. coli and K. pneumoniae, 32 and 8 µg/mL respectively, it was

not considered suitable as a clinical candidate itself. NOSO-95179 was

selected as a starting point in a lead optimization program with a ﬁrst

objective of improving its in vitro antibacterial activity against E. coli

and K. pneumoniae.

New classes of antibacterial compounds are urgently needed as

molecules currently in clinical development are mainly derivatives of

known classes that might not overcome the existing resistance

The antibacterial activity of a compound is governed by the activity

^⁎Corresponding authors.

E-mail addresses: edgars@osi.lv (E. Suna), e.racine@nosopharm.com (E. Racine).

https://doi.org/10.1016/j.bmc.2020.115469

Received 11 October 2019; Received in revised form 10 March 2020; Accepted 25 March 2020

0968 -0 896 /©202 0 E l s e v i e r L t d .A l l r i g h t s r e s e r v ed.

Pleasecitethisarticleas:EinarsLoza,etal.,Bioorganic&MedicinalChemistry,https://doi.org/10.1016/j.bmc.2020.115469

E. Loza, et al.

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Fig. 1. Structure of NOSO-95179 1 and positions of SAR studies.

on target, by the crossing of the bacterial membranes in the case of

intracellular targets, as for NOSO-95179, and by the propensity of a

compound to evade eﬄux. In the case of NOSO-95179, it was pre-

viously determined that eﬄux activity in E. coli was not a primary

determinant of the antibacterial activity, as the MIC against an E. coli

strain with deletion in the outer membrane component of the main

eﬄux pumps found in E. coli (ΔtolC) was the same as the MIC on wild-

type E. coli (8 µg/mL).¹²Regarding the crossing of the bacterial mem-

branes of Gram-negative bacteria, so far there is a lack of understanding

of the molecular basis for compound penetration into these bacteria and

despite compilation and analysis of physicochemical properties of

known antibacterial compounds, it is challenging to apply the observed

trends to a new family of compounds.^13,14,15On top of this, the lack of

tools to measure compound penetration into Gram-negative bacteria

has been identiﬁed as a key bottleneck for the rational discovery of

novel antibacterial compounds.¹⁶Based on this, ﬁrst eﬀorts to optimize

NOSO-95179 were directed towards the improvement of the inhibition

of bacterial translation. By the time this study was started, bacterial

ribosome was identiﬁed as the target for ODLs but co-crystallisation

studies were not available to do rational structure-based drug design.

From previous structure–activity relationship (SAR) studies on one

of the ﬁrst Odilorhabdin isolated (NOSO-95C), it was known that amino

acids of positions 2 (Dab(βOH)) and 5 (^D-Orn) are key for the inhibition

of bacterial translation, as replacing them with alanine led to a strong

decrease of the inhibition, and as a consequence to a loss of anti-

bacterial activity.¹¹We postulated that these moieties were key for

compound binding to the bacterial ribosome and that they could be

replaced by structures that would improve even further the binding to it

and as such the inhibition of the translation. Thus, amino acids with

structures closer to Dab(βOH)2 and D-Orn5 than alanine were con-

sidered, ﬁrst using commercially available amino acids. As the struc-

tures of commercially available amino acids are quite diﬀerent from the

original Dab(βOH) and ^D-Orn, the synthesis of closely related structures

and their incorporation into NOSO-95179 was planned. The strategy of

replacing non-proteinogenic amino acids to improve antibacterial eﬃ-

cacy of antimicrobial peptides (AMPs) has been described before.

Teixobactin is a cyclodepsipeptide that bears ﬁve non-proteinogenic

amino acids such as ^L-allo-enduracididine. Some of the analogues in

which this amino acid is replaced by L-arginine^17,18or L-lysine¹⁹dis-

play an activity similar to that of teixobactin. Daptomycin, another

potent cyclodepsipeptide, bears the non-proteinogenic amino acids

(2S,3R)mGlu in position 12 and kynurenine in position 13. An analogue

of daptomycin bearing a glutamic acid in position 12 and a tryptophan

in position 13 displays antibacterial eﬃcacies similar to daptomycin

against two strains of Bacillus subtilus.²⁰

synthesis of NOSO-95179 analogues. Inhibition of bacterial translation

of each of the synthesized Odilorhabdin analogues was measured using

an in vitro test. For the most eﬃcient analogues, antibacterial eﬃcacy

was evaluated against two wild-type Enterobacteriaceae (E. coli and K.

pneumoniae) and against an eﬄux-defective E. coli strain (ΔtolC) to

evaluate the impact of eﬄux on the antibacterial activity. Interesting

structure–activity relationships were found through this study that will

help in the design of future analogues.

2. Results and discussion

2.1. Synthesis of amino acids

Structure-activity relationship studies in NOSO-95179 1 required a

versatile synthetic approach to a series of previously unreported ana-

logues of (2S,3S)-α,χ-diamino β-hydroxy butyric acid (Dab(βOH)) and

D–ornithine (Fig. 1). Ideally, the synthetic strategy should provide ac-

cess to both L - and

purity. In addition, a ^Dd^-e^ss^ei^rrⁱa^eb^sle^offe^aa^mtuⁱrⁿe^oof^acth^ide^ss^wyn^itt^hhe^hsiⁱs^ghap^epⁿro^aaⁿc^tih^omis^et^rhⁱe^c

ability to increase the optical purity of products by chromatography or

crystallization. We realized that Williams’ chiral diphenyloxazinone²¹is

perfectly suited as a building block for the diastereoselective synthesis

of both Dab(βOH) and ^D–ornithine analogues. Thus, the former could be

accessible by an asymmetric aldol-type reaction of the diphenylox-

azinone-derived boron enolate with suitable aldehydes, whereas ^D-or-

nithine analogues could be synthesized by diastereoselective alkylation

of the corresponding sodium enolate (Fig. 2).

Anti-diastereoselective aldol reaction between N-Boc protected

aminoaldehydes 3a–e and boron enolate of Williams’ chiral dipheny-

loxazinone (S,R)–4 was the key step in the synthesis of Dab(β OH) de-

rivatives 8a–d, 9b and β-hydroxyornithine 8e (Scheme 1).^22,23,24Al-

dehydes 3a,d,e were prepared by oxidation of the corresponding amino

alcohols 2a,d,e by Dess-Martin periodinane, whereas aldehydes 3b,c

are commercially available. The boron enolate was obtained in situ from

diphenyloxazinone (S,R)–4 and Bu₂BOTf in the presence of Et₃N at

−78 °C. The aldol reaction between the boron enolate of (S,R)–4 and

aldehydes 3a–e proceeded with good diastereoselectivity (> 4:1 d.r.).

The desired major diastereomers were obtained in 28–56% yield after

crystallization or silica gel chromatography. It has been reported that

the Bu₂BOTf-mediated aldol reaction between diphenyloxazinone

(S,R)–4 and Cbz-protected analog of 3e provided the anti-aldol product

N^ω-Cbz-6e with the 2S,3S conﬁguration of the newly created stereo-

genic centers.²³By analogy, the structurally closely related aldol pro-

ducts 6a–e were also assigned 2S,3S conﬁguration. Cleavage of both the

chiral auxiliary and N-Cbz protecting group in (2S,3S)-6a,b,d,e and

(2S,3R)-6c by Pd-catalyzed hydrogenation aﬀorded amino acids

(2S,3R)-7a–c and (2S,3S)-7d–e, which were converted into N^α-Fmoc

In this paper we describe the synthesis of previously unreported

non-proteinogenic amino acids and their use in the solid phase peptide

2

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Fig. 2. Synthetic approach to Dab(βOH) and D-Orn analogues.

derivatives (2S,3S)-8a,b,d,e and (2S,3R)-8c by reaction with FmocOSu.

Amino acid (2S,3S)-8b was elaborated into acetonide (2S,3R)-9b by

reaction with 2,2-dimetoxypropane in the presence of boron triﬂuoride

etherate.

respect to the C-2 methyl. This ﬁnding is corroborated by an NOE be-

tween a proton of the C-3 hydroxyl group and the benzylic proton of

Williams’ chiral auxiliary. Furthermore, a large coupling constant (³J_1H-

_13C) of 13.5 Hz between the C-3 proton and the carbon of the acid

moiety indicated an antiperiplanar relationship between these nuclei.

These data provide strong support for the 2S,3S stereochemical as-

signments in the aldol product (2S,3S)–6f.

Synthesis of the α-quaternary center-containing amino acid (2S,3S)-

8f required an initial alkylation of the sodium enolate of chiral diphe-

nyloxazinone (S,R)–4 with MeI to furnish (2S)(S,R)–5 in excellent

diastereoselectivity (99:1 dr).²⁵Subsequent treatment of (2S)(S,R)–5

with Bu₂BOTf and Et₃N at −78 °C aﬀorded the corresponding boron

enolate. Surprisingly, no conversion was observed in the reaction be-

tween the boron enolate of 5 and aldehyde 3f under the standard aldol

reaction conditions.^23,26The use of the more reactive sodium enolate of

5, which aﬀorded a complex mixture of products, was also un-

successful. Finally, it was found that the desired aldol product 6f could

be obtained under Mukaiyama aldol conditions.²⁷Accordingly, the in-

itial conversion of (2S)(S,R)–5 into the O-TBS-protected enol ether was

followed by a TBAF-promoted aldol reaction with 3f to aﬀord the de-

sired 6f as a 83:17 mixture of diastereomers. Diastereomerically pure 6f

(> 99:1 d.r.) was obtained in 38% yield after puriﬁcation by column

chromatography. The relative conﬁguration of the newly created qua-

ternary center in the aldol product 6f was assigned 2S based on the

well-established stereoinduction model for aldol reactions involving

Williams’ chiral auxiliary.^21,22,23Accordingly, an aldehyde approaches

the diphenyloxazinone-derived E-enolate from the face opposite to the

two phenyl rings. Support for the anti-relationship between the newly

created stereogenic centers C-2 and C-3 in 6f was obtained by NMR

experiments (Fig. 3). Thus, strong nuclear Overhauser eﬀects (NOEs)

were observed between the C-2 methyl group and protons at C-3 and C-

4 indicating that the C-3 hydroxyl group is in anti-conformation with

The synthesis of the Dab(βOH) diastereoisomer (2S,3R)–8g required

a syn-selective aldol reaction between the diphenyloxazinone (S,R)–4

and the aldehyde 3f to achieve an opposite diastereoselectivity to that

of the Mukayiama aldol conditions (used for (2S,3S)–6f) and the aldol

reaction involving boron enolate (employed for (2S,3S)–6a-e). In the

latter case, the anti-selectivity was proposed to result from the addition

of the boron enolate to the Re face of the aldehyde in the chair-like

conformation of the six-centers closed transition state, with the alde-

hyde substituent occupying equatorial position (see TS-1 in Fig. 3).²²

We realized that the desired syn-selectivity would require addition of

the enolate from the Si face of the aldehyde. This implies axial or-

ientation of the aldehyde substituent in the cyclic transition state for the

aldol reaction. We hypothesized that the desired syn-selective cyclic

transition state could be achieved by chelation of the enolate metal ion

by the Lewis-basic carbamate subunit of the aldehyde (see TS-2, Fig. 3).

After extensive experimentation (results not shown) we found that the

use of the zinc enolate of diphenyloxazinone (S,R)–4 eﬀected the de-

sired syn-diastereoselectivity in the reaction with aldehyde 3f (5:1 sy-

n:anti ratio). The major syn-diastereomer (2S,3R)–6g was obtained in a

pure form after puriﬁcation by silica gel column chromatography.

Subsequent Pd-catalyzed hydrogenation aﬀorded the amino acid

(2S,3R)–7g, which was reacted with FmocOSu to provide (2S,3R)–8g

Scheme 1. Synthesis of Dab(βOH) derivatives 8a–g and 9b. Reagents and conditions: (a) Dess-Martin periodinane (1.2–1.4 equiv.), DCM, rt, 77–94%. (b) NaHMDS

(1.5 equiv.), THF, then MeI (6 equiv.), −78 °C, 1 h, 84%. (c) 3a-e (1.8 equiv), (R,S)–4 (1 equiv), Bu₂BOTf (2 equiv), TEA, DCM, –78 °C, 1 h, 28–56%. (d) (2S,S,R)–5,

NaHMDS (1.05 equiv), TBSCl, THF, –78 °C, 1 h, 57%, then 3f, TBAF, THF −78 °C, 1 h, 38%. (e) (S,R)–4, NaHMDS (1 equiv), ZnBr₂(1 equiv), THF, then 3f (0.5

equiv), −78 °C, 1 h, 26%. (f) H₂(1 atm), 10% Pd/C, MeOH/THF, rt, 2 h, 44–98% (crude). (g) for 8a-e, g: FmocOSu (1–1.5 equiv.), aq. NaHCO₃, dioxane, rt,

overnight, 48–73%; for 8f: FmocOSu (1.5 equiv.), FmocCl (1.4 equiv.), aq. NaHCO₃, dioxane, rt, 42 h, 23%. (h) Me₂C(OMe)₂(23 equiv.), BF₃.Et₂O (0.12 equiv),

acetone, rt, 3 h, 80%.

3

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Fig. 3. Structure assignment for 6f and the proposed transition states for the aldol reaction between 3f and boron or zinc enolates of (S,R)-4.

Scheme 2. Synthesis of ornithine derivatives 16a-c and amino acid 21. Reagents and conditions: (a) NaHMDS (1.3 equiv.), THF, then MeI (5 equiv.), −78 °C, 1 h,

65%. (b) PhCHO (1 equiv.), aq. NaOH, MeOH, rt, 1 h, then NaBH₄(0.4–1 equiv.), 0 °C to rt, 1 h, crude (11a), 90% (11b). (c) for 11a: Boc₂O (1.04 equiv.), TEA, water,

dioxane, 0 °C, 18 h (68% from 10a); for 11b: Boc₂O (1.05 equiv.), TEA, DCM, rt, 18 h (80%); for 11c: Boc₂O (1 equiv.), 5:1 DCM:NaOH (1 M aqueous solution), rt,

16 h, (66%). (d) BH₃–THF (3 equiv.), THF, −72 °C → rt, 20 h, 88%. (e) Ph₃P (1.3 equiv.), I₂(1.33 equiv.), imidazole (1.75 equiv.), DCM, rt, 1 h, 45% (13a), 38%

(13b) and 83% (13c). (f) (R,S)–4 (1 equiv.), NaHMDS (1.1 equiv.), HMPA, THF, −75 °C, 10 min, then 13a,b or 18 (1.3 equiv.), –55 °C, 2 h, 72% (14a), 58% (14b),

23% (19). (g) (2R)(R,S)–5 (1 equiv.), KHMDS (1.3 equiv.), 13c (2.2 equiv.), HMPA, THF, −78 °C, 4 h, 82%. (h) Li (27–30 equiv.)/NH₃, THF, −75 °C, 1 h, 29% (15c);

products 15a,b were used in the next step in a crude form. (i) FmocOSu (1–3 equiv.), aqueous NaHCO₃, dioxane, rt, 36 h, 22% (16a, from 14a), 27% (16b, from 14b),

76% (16c) and 44% (21). (j) NaI (1.25 equiv.), acetone, rt, 24 h, 69%. (k) Pd-C (10%) (0.3 equiv.), H₂(1 atm), MeOH/THF, rt, 2 h, 74%.

(Scheme 1). The stereochemical assignment of the stereogenic C-2 and

C-3 centers of (2S,3R)–8g was further conﬁrmed by comparison of NMR

spectra of (2S,3R)–8g with that of the diastereomeric (2S,3S)–8g, which

was obtained by a diﬀerent synthetic route.¹¹

reductive cleavage of both Williams’ chiral auxiliary and N-Bn group by

lithium in liquid ammonia.³⁰The formed amino acids (2R)–15a-c were

converted into the target N^α-Fmoc derivatives (2R)–16a-c in the reaction

with FmocOSu in the presence of aqueous NaHCO₃(Scheme 2). The

diastereoselective alkylation of the sodium enolate of Williams’ chiral

diphenyloxazinone (R,S)–4 was also employed for synthesis of the 5-

hydroxynorvaline derivative 21. Alkylating reagent 18 was prepared

from commercially available bromide 17 under Finkelstein’s reaction

conditions. The highly diastereoselective (99:1 d.r.) alkylation step was

followed by a hydrogenation/Fmoc protection sequence to furnish the

target amino acid 21 (Scheme 2).

The key step in the synthesis of ornithine analogues 16a-c was al-

kylation of the sodium enolate of Williams’ chiral diphenyloxazinone

(R,S)-4 and the potassium enolate of its methyl analog (2R)(R,S)-5 with

the alkyl iodides 13a,b and 13c, respectively (Scheme 2). Alkylating

reagents 13a,b were synthesized from commercially available (S)-3-

aminobutanoic acid 10a and (R)-3-aminobutan-1-ol 10b. The synthesis

comprised N-benzylation of 10a–c under reductive amination conditions,

followed by N-Boc protection (and borane reduction of the acid moiety in

N-Boc-11a) to aﬀord alcohols 12a–c, which were elaborated into iodides

13a-c under Appel’s reaction conditions. The alkylation of sodium en-

olate of (R,S)–4 with iodides 13a,b and potassium enolate of (2R)(R,S)-5

with 13c proceeded with excellent diastereoselectivity (99:1 d.r.). The

newly formed α-stereogenic center in 14a-c was assigned the 2R con-

ﬁguration based on the well-described anti-diastereoselectivity of the

alkylation, with the alkylating agent approaching the enolate of Wil-

liams’ chiral glycine equivalent from the less hindered face opposite to

the phenyl substituents.^25,28,29Final steps in the synthesis involved the

(2S,3S)–2,4-Diamino-3-methoxybutanoic acid 27 was synthesized

from 5-hydroxyectoin via the previously reported diastereomerically

pure copper complex (2S,3S)–22 (Scheme 3).¹¹Hydrolysis of complex 22

with NaOH and EDTA was followed by N -Cbz protection of the α-amino

group using Cbz-OSu to aﬀord (2S,3S)–23. Chemoselective alkylation of

carboxylate moiety with BnBr furnished the ester (2S,3S)–24, which was

converted into the methoxy-derivative (2S,3S)–25 by alkylation with MeI

in the presence of silver(I) oxide. Subsequent deprotection of both the

acid moiety and the α-amino group under catalytic hydrogenation con-

ditions provided the amino acid (2S,3S)–26. The synthesis was

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Scheme 3. Synthesis of Dab(βOH) derivative (2S,3S)–27 and ornithine analogue (R)-31. Reagents and conditions: (ref. 8) i: NaOH (2 equiv.), H₂O, 50 °C, 6 h

followed by aq. 6 N HCl, 110 °C, 3 h. ii: NaOH (3 equiv.), CuSO₄·5H₂O (0.5 equiv.), 110 °C followed by Boc₂O (2.5 equiv.), dioxane, rt, 96 h, 40% (from 5-

hydroxyectoin). (a) NaOH, Na₂EDTA·2H₂O (1.5 equiv.), H₂O, rt, 4 h; then CbzOSu (0.95 equiv.), Na₂CO₃, dioxane, rt, 16 h, 88%. (b) BnBr (1.1 equiv.), DIPEA, DMF,

rt, 48 h, 95%. (c) Ag₂O (5 equiv.), MeI (9.9 equiv.), MeCN, rt, 3 d, crude product used in the next step. (d) H₂, Pd-C (10%) (0.1 equiv.), MeOH, rt, 1 h, 41% (from 24).

(e) FmocOSu (1 equiv.), aq. NaHCO₃, dioxane, rt, 36 h, 69%. (f) paraformaldehyde (2 equiv.), CSA (0.08 equiv.), MeCN, microwaves, 120 °C, 2 min, 73%. (g) Et₃SiH

(6 equiv.), TFA (6 equiv.), Cl(CH₂)₂Cl, 60 °C, 18 h, crude. (h) Boc₂O (1 equiv.), NaHCO₃, H₂O, THF, rt, 18 h, 62%.

completed by N^α-Fmoc protection of the amino group to aﬀord the target

(2S,3S)–27 (Scheme 3). The synthesis of protected N^δ-monomethyl-D-

ornithine 31 was accomplished by conversion of the amino acid 28 into

the cyclic aminal 29, followed by regioselective reductive ring opening to

30³¹and N-Boc protection (Scheme 3).

mechanism of action of ODLs, including NOSO-95179, was identiﬁed as

inhibition of the bacterial translation through binding to the bacterial

ribosome based on macromolecular synthesis assay, resistant mutants

selection and whole genome sequencing.³⁴Inhibition of bacterial

translation by NOSO-95179 1 and novel analogues was measured using

an in vitro coupled transcription-translation (IVTT) system. Using a cell-

free protein synthesis system from E. coli, the synthesis of a ﬂuorescent

protein was followed by measuring ﬂuorescence. This assay has been

described before as useful to rank compounds with regard to inhibition

of transcription or translation.³⁵The IC₅₀of NOSO-95179 1 was mea-

sured at 0.55 µM. This value is in the range of other translation in-

hibitors like gentamicin (0.31 µM), chloramphenicol (7.74 µM), spec-

tinomycin (0.99 µM) and erythromycin (2.12 µM).

The key step in the synthesis of amino acid (2S,3S)–39 was the

diastereoselective Michael addition reaction between the chiral Ni(II)

complex (S)–32³²and the (E)–crotonic acid methyl ester (Scheme 4).³³

The Michael addition product (2S,3S)–33 was formed with 70:30 d.r.,

however the major diastereoisomer was obtained with 95:5 d.r. by se-

lective precipitation from the reaction mixture upon addition of 5%

aqueous AcOH. Assignment of an absolute conﬁguration for the newly

created stereogenic centers in (2S,3S)–33 was based on the analogy

with that in the Michael adduct of (S)–32 to ethyl (E)–crotonate.³²

Hydrolysis of the ester moiety under basic conditions aﬀorded acid

(2S,3S)–34, which was converted into the azide (2S,3S)–35 via an in-

termediate mixed anhydride (Scheme 4). Crude azide (2S,3S)–35 was

subjected to Curtius rearrangement in the presence of allylic alcohol to

furnish the N-Alloc-protected Ni(II) complex (2S,3S)–36 (95:5 d.r.).

Acidic hydrolysis of the Ni(II) complex was followed by N-Fmoc pro-

tection of the α-amino group to aﬀord the amino acid (2S,3S)–37. The

end-game of the synthesis included the change of the N^γ-Alloc pro-

tecting group for N^γ-Boc to yield the target Dab(βOH) derivative

(2S,3S)–39 as a single diastereomer (99% d.e.) after puriﬁcation by

chromatography (Scheme 4).

Modiﬁcation of the (2S,3S)-α,χ-diamino β-hydroxy butyric acid

(Dab(βOH)) in position 2 was investigated (Table 1). First, the inﬂuence

of the amine function was studied by removing it. Replacing Dab(βOH)

by AlloThr (40) or Ser (41) led to analogues displaying a strong de-

crease of the inhibition of the translation. The inﬂuence of the hydroxyl

function was then studied by removing it. The analogue bearing a 2,4-

diamino butyric acid (Dab, 42) was synthesized and evaluated. In that

case, the IC₅₀of the inhibition of bacterial translation was slightly

better than for analogues 40 and 41 bearing only a hydroxyl function

(17.0 µM vs > 40 µM) but 34-fold worse than for NOSO-95179 1

bearing Dab(βOH). Based on these ﬁrst results the presence of both the

amine and the hydroxyl functions is important for the inhibition of

bacterial translation. Thus, analogues bearing amino acids closely re-

lated to Dab(βOH) in which both functions are kept or only slightly

modiﬁed were synthesized and evaluated. The ﬁrst analogues were

designed to replace, mask or change the stereochemistry of the hy-

droxyl function. When both the H-bond donor and acceptor properties

of the hydroxyl function were removed by replacing it by a methyl (43)

a 50-fold decrease of the inhibition of bacterial translation was ob-

served. Removing the H-bond donor properties while keeping the ac-

ceptor properties through replacement of the hydroxyl function by a

With these original amino acids in hand, analogues of NOSO-95179

in positions 2 and 5 were synthesized and evaluated for their potency to

inhibit bacterial translation.

2.2. Structure-activity relationships study on positions 2 and 5 of NOSO-

95179

All the analogues were synthesized via SPPS following a method

described previously.¹¹By the time this study was performed, the

5

E. Loza, et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

Scheme 4. Synthesis of 3-methyl Dab(βOH) derivative (2S,3S)–39. Reagents and conditions: (a) Methyl (E)–crotonate (1.1 equiv.), DBU (0.5 equiv.), DMF, rt, 60 h,

52%. (b) NaOH (6 equiv.), H₂O, EtOH, rt, 1 h, 88%. (c) ClCO₂Et (3 equiv.), TEA (4 equiv.), THF, rt, 30 min, then NaN₃(5 equiv.), H₂O, rt, 2 h. (d) CH₂=CHCH₂OH,

toluene, 80 °C, 16 h, 66% (from 34). (e) Aqueous conc. HCl, MeOH, 60 °C, 4 h, then FmocOSu (1.5 equiv.), NaHCO₃, H₂O, dioxane, rt, 64 h, 51%. (e) PdCl₂(PPh₃)₂

(0.02 equiv.), n-Bu₃SnH (2.1 equiv.), H₂O, DCM, rt, 6 h, 84%. (f) Boc₂O (2 equiv.), NaHCO₃, H₂O, THF, rt, 18 h, 74%.

methoxy group (44), or reversing its stereochemistry (45), led to even a

greater loss of the inhibition of translation (> 80-fold decrease for both

analogues compared to NOSO-95179). Based on these results, no more

modiﬁcations of the hydroxyl function were evaluated, and the design

of next analogues focused on modifying the amine function and the

lateral chain. Modiﬁcation of the position of the amine function was

ﬁrst investigated by increasing the chain length between the hydroxyl

and the amine moieties (46). Again, the inhibition of bacterial trans-

lation was lowered by 30-fold. Next, the carbon in the alpha position of

the amine function was substituted with one or two methyl groups with

the aim of forcing the amine moiety to adopt a favourable position for

the inhibition of bacterial translation (47, 48, 49). A strong increase of

the IC₅₀was observed for analogues bearing a (4S)-Me and a dimethyl

substitution (47 and 49, IC₅₀> 80-fold compared to NOSO-95179)

while the analogue bearing a (4R)-Me substitution (48) showed a

slightly better inhibition (IC₅₀20-fold higher compared to NOSO-

95179). Finally, the amine function was methylated to increase its

basicity (50).³⁶In that case, the inhibition of bacterial translation was

closer to the one measured for NOSO-95179 (3.2 µM vs 0.5 µM).

alpha centre (59) were introduced to force the amine function into a

suitable position. This strategy was not eﬃcient in improving the in-

hibition of bacterial translation. In view of these results, substitutions in

the β and χ positions of the lateral chain were not further investigated.

MICs values of analogues showing inhibition of bacterial translation

close to NOSO-95179 were measured against two wild-type

Enterobacteriaceae (E. coli ATCC 25,922 and K. pneumoniae ATCC

13883), and against an eﬄux-defective (ΔtolC) E. coli strain isogenic to

ATCC 25,922 to evaluate the inﬂuence of eﬄux on the antibacterial

activity (Table 3). Antibacterial activities of analogue 50 on the three

strains tested were 8-fold higher than the MIC of NOSO-95179 which is

consistent with a 6-fold decrease of the inhibition of translation. The

higher steric hindrance and/or basicity brought by the methyl sub-

stitution of the amine function of the lateral chain of Dab(βOH) doesn’t

render this analogue more susceptible to eﬄux. For analogue 53, the

antibacterial activity was strongly decreased on the three strains despite

the modest 2.5-fold increase of the IC₅₀. One hypothesis to explain the

low antibacterial activity could be that replacing the amine function of

the lateral chain of ^D-Orn by a hydroxyl function decreases strongly the

ability to cross one or both of the bacterial membranes. This hypothesis

would be consistent with the observations made by Richter et al.

showing that the presence of primary amine helps crossing the bacterial

membranes.³⁷

Modiﬁcations of ^D-Orn in position

5 were then investigated

(Table 2). The ﬁrst study aimed at increasing or decreasing the length of

the lateral chain. When ^D-Orn was replaced by 2,4-diaminobutyric acid

(^D-Dab, 51) or by D-Lys (52) the inhibition of bacterial translation was

strongly reduced for both analogues (> 80-fold). Keeping or removing

the H-bond donor and acceptor properties of the amine function by

replacing it respectively by a hydroxyl function (53) or by a methyl

group (54) led to interesting results. For the analogue bearing a hy-

droxyl function (53), IC₅₀was close to the one measured for NOSO-

95179 (1.3 µM versus 0.5 µM) while for the analogue bearing a methyl

(54) IC₅₀was increased by > 80-fold. Having a function with H-donor

and H-acceptor properties in this position is key for the inhibition of

bacterial translation. The basicity of the amine function was then im-

proved by substituting it by a methyl (55)³⁶or decreased via substitu-

tion by an acetyl group (56). In both cases the inhibition of bacterial

translation was strongly decreased. Regarding analogue 55, it could be

that the steric hindrance caused by the methyl substitution disturbs

binding to the target. Finally, increased steric constraints through in-

corporation of a methyl group on the lateral chain (57, 58) or at the

In this study, the two positions of the lead compound NOSO-95179

previously identiﬁed as key for the inhibition of bacterial translation

were modiﬁed using both commercially available amino acids and

newly synthesized ones. The objective was to identify moieties that

would improve even further the binding to the bacterial ribosome and

as such improve the antibacterial activity of the new analogues. Two

analogues showed IC₅₀for the inhibition of the bacterial translation in

the range of NOSO-95179 but the in vitro antibacterial activities as

measured by MIC were decreased. For one analogue in which the amine

function of lateral chain of D-Orn5 was replaced by a hydroxyl function,

the antibacterial activities were strongly decreased compare to NOSO-

95179 while their IC₅₀for the inhibition of the translation were very

similar. This could be due to a decreased permeation across bacterial

membranes.

A few months after this study, co-crystallization studies between

6

E. Loza, et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

Table 1

Table 2

Modiﬁcation of Dab(βOH) in position 2 of NOSO-95179 1. SD: standard de-

Modiﬁcation of ^D-Orn in position 5 of NOSO-95179 1. SD: standard deviation. ^a

viation.

Single experiment.

Compound

1

R1

R2

IC₅₀

(

SD, µM)

0.1

H

0.5

Compound

1

R

IC₅₀

(

SD, µM)

0.5

0.1

51

52

H

36.3

0.3

40

41

42

> 40

17.0

> 40

1.3^a

7.2

5.5

53

H

43

44

45

46

26.7

> 40

14.4

54

55

H

> 40

3.2

56

H

> 40

47

48

49

50

> 40

10.0

> 40

3.2

57

58

59

H

17.9

0.8

0.3

0.1

H

> 40

18.1^a

Me

NOSO-95179 and the ribosome of the bacterial specie Thermus ther-

mophilus conﬁrmed the importance of these two moieties for binding to

the target.⁸Both the hydroxyl and amine functions of Dab(βOH) and

the amine function of the lateral chain of ^D-Orn form hydrogen bonds

with sugar-phosphate backbone atoms of 16S rRNA of the bacterial

ribosome. As expected based on co-crystallisation, for analogues in

which Dab(βOH) is replaced by amino acids bearing only the hydroxyl

function (40 and 41) or the amine function (42), inhibition of bacterial

translation was strongly reduced and could not be measured high-

lighting the importance of having these two functions for an eﬃcient

binding to the target. Of analogues bearing both the amine and the

hydroxyl function, analogue 50 in which the amine function is me-

thylated displayed the best inhibition of the bacterial translation.

Docking studies of analogue 50 and NOSO-95179 1 in the bacterial

Table 3

In vitro antibacterial activity of analogues showing the best inhibition of

translation^.IBT: inhibition of the bacterial translation; MIC: minimum in-

hibitory concentration; WT: wild-type; E.c. WT: Escherichia coli ATCC 25922,

E.c. ΔtolC: eﬄux-defective (ΔtolC) Escherichia coli strain isogenic to ATCC

25922 K.p. WT: Klebsiella pneumoniae ATCC 13883.

Compound

IC₅₀IBT (µM)

MIC (µg/mL)

E.c. WT

E. c. ΔtolC

K.p. WT

1

0.5

3.2

1.3

8

4

50

53

64

32

> 64

7

E. Loza, et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

ribosome have been performed (cf Supporting Information).⁸Distance for

the hydrogen bonding between the hydroxyl function of 50 and NOSO-

95179 was the same (2.7 Å) but the bond has moved from oxygen of

phosphate (P]O) between 16S residues C1051 and U1052 for NOSO-

95179 to the one between 16S residues G1050 and C1051 for analogue

50. While the amine function of NOSO-95179 made hydrogen bond

only with oxygen of phosphate (P]O) between 16S residues G1050 and

C1051 (3.8 Å), the methylated amine of analogue 50 made hydrogen

bonds with the same oxygen than NOSO-95179 (5.0 Å) but also with

oxygen of phosphate (P]O) between 16S residues U-1199 and C-1200

(3.3 Å). Regarding modiﬁcation of ^D-Orn, only the analogue bearing a

hydroxyl function instead of the amine function (53) displayed an in-

hibition of bacterial translation similar in magnitude to that of NOSO-

95179. This result can be explained by the hydroxyl group still being

4. Materials and methods

4.1. General Information

4.1.1. Synthesis of non-proteinogenic amino acids

Nuclear magnetic resonance spectra were recorded on NMR spec-

trometers at the following frequencies: ¹H, 400 or 300 MHz, proton-

decoupled ¹³C, 101 MHz. Chemical shifts are reported in parts per

million and referenced to the residual solvent signal. High-resolution

mass spectra (HRMS) were recorded on a time-of-ﬂight (TOF) mass

analyzer using ESI technique. The purity of the ﬁnal compounds was

conﬁrmed to be ≥ 95% with HPLC systems equipped with separation

module with quaternary pump, degasser, autosampler, column ther-

mostat, and dual absorbance detector. Analytical thin-layer chromato-

graphy (TLC) was performed on pre-coated silica gel F-254 plates.

Chromatographical puriﬁcations of compounds were performed by

column chromatography using silica gel (particle size 0.035–0.070 mm) or

pre-packed columns with C18-silica gel (particle size 0.040–0.063 mm).

Chemicals and reagents were obtained from commercial suppliers and

were used without further puriﬁcation. Anhydrous THF, Et₂O and di-

chloromethane (DCM) were obtained by passing commercially available

solvents through activated alumina columns.

able to form

a hydrogen bond with sugar-phosphate backbone

atoms of bacterial RNA. Docking studies of this analogue and NOSO-

95179 1 in the bacterial ribosome have been performed (cf Supporting

Information).⁸The amine function of the lateral chain of D-Orn of NOSO-

95179 and the hydroxyl function of the lateral chain of analogue 53

formed hydrogen bonds with sugar-phosphate backbone atoms of 16S

rRNA. The distance for the hydrogen bonding with ribose oxygen O4′ of

16S rRNA residue C1214 was shorter for the hydroxyl function as

compared to that for the amine function (2.8 and 4.2 Å respectively). In

contrast, the distance with oxygen of phosphate (P]O) between 16S

residues C1214 and G1215 was shorter for the amine function than for

the hydroxyl function (3.1 and 4.8 Å respectively). In the case of ana-

logues still bearing a primary amine function on the lateral chain of

position 5 (51, 52, 57, 58 and 59) the low inhibition of bacterial

translation could be explained by a position of the amine function not

favourable to form an hydrogen bond with the oxygen of the bacterial

RNA. In the case of analogue 55 bearing a secondary amine, and 56

bearing an amide, steric hindrance brought by the methyl or the acyl

function could disfavour the binding between these analogues and the

bacterial ribosome. For analogue 54, absence of H-bond donor function

could explain its low activity.

4.1.2. Peptide synthesis

All commercially available reagents were used without further pur-

iﬁcation unless otherwise noted. Resins, HBTU and protected amino

acids were purchased from Chem-Impex International Inc (Wooddale, IL,

USA). All other reagents and solvents, including of HPLC grade, were

purchased from Sigma-Aldrich (St Louis, MO, USA).

Puriﬁcation of peptides was performed on a HPLC Agilent 1260

Inﬁnity system with a Waters Symmetry semi preparative C18 column

(7 µm, 7.8 mm × 300 mm). 50 µL of peptide solution (300 mg/mL)

were injected, the ﬂow rate was set up at 2.5 mL/min and UV detection

was made at 230 nm. The following mobile phases were used: A: 0.1%

triﬂuoroacetic acid in water; B: acetonitrile. The following gradient was

used: 0 to 15% of B in A from 0 to 15 min.

Structure-based drug design using co-crystallization data could help

designing next-generation analogues with a better aﬃnity for the bac-

terial ribosome. This strategy has been used before by Melinta

Therapeutics (previously Rib-X Pharmaceuticals) to develop an anti-

bacterial program starting from two natural products that bind to the

bacterial ribosome (Blasticidin S and TAN-1057).^38,39Based on the

crystal structure of Blasticidin S bound to the Haloarcula marismortui

ribosome, two Blasticidin S ligands bound to the ribosome were iden-

tiﬁed. Using structure-based drug design, various active analogues were

obtained. An additional strategy to ﬁnd more potent Odilorhabdins’

analogues would be to design compounds having an additional inter-

action with the ribosome.

Analytical LC-MS of peptides was performed on the same system with

a Waters Symmetry analytical C18 column (5 µm, 4.6 mm × 150 mm).

10 µL were injected, the ﬂow rate was set up at 0.7 mL/min and UV

detection was made at 230 nm. The following methods were used:

method A (mobile phases: A: 0.1% triﬂuoroacetic acid in water; B:

acetonitrile; gradient: 2 to 30% of B in A from 0 to 15 min); method B

(mobile phases: A: 0.2% heptaﬂuorobutyric acid in water; B: acetonitrile;

gradient: 20 to 50% of B in A from 0 to 15 min). ESI-LC-MS data were

obtained in the positive mode on an Agilent 1260 Inﬁnity system

(Agilent 6120 Quadrupole LC/MS, 1260 Quaternary Pump, 1260 ALS,

1260 TCC, 1260 DAD VL, 1260 FC-AS).

4.2. General procedure for solid-phase peptide synthesis (SPPS)

3. Conclusion

All reactions were carried out in polypropylene empty reservoirs

(15 mL) ﬁtted with polyethylene frits and polytetraﬂuoroethylene

stopcock at room temperature.

The objective of this study was to ﬁnd analogues of the ﬁrst

Odilorhabdin lead compound (NOSO-95179) displaying improved in-

hibition of the bacterial translation. The synthesis of thirteen previously

unreported non-proteinogenic amino acids was developed mainly based

on the highly diastereoselective alkylation and aldol-type reaction of

Williams’ chiral diphenyloxazinone. Twenty analogues of NOSO-95179

were synthesized by solid-phase peptide synthesis. Important informa-

tion regarding the SAR of Odilorhabdins was found that could help in

the design of future analogues despite the lower inhibition of bacterial

translation of the analogues synthesized and described here. Future

studies to improve both in vitro activity and in vivo eﬃcacy of NOSO-

95179 will focus on structure-based drug design, improvement of

membrane permeation ability, and improvement of in vivo exposure of

NOSO-95179 through, for instance, improving its metabolic stability

and slowing down its rate of elimination.

Loading of ﬁrst amino acid: 2-chlorotrityl chloride resin (75 mg,

1.0 mmol/g) was swelled in DCM (3.0 mL) for 30 min. DCM was re-

moved then a solution of Fmoc-protected amino acid (3.0 equiv.) and

DIPEA (4.0 equiv.) in DCM (1.3 mL) was added. The reservoir was

shaken for 240 min, the reaction mixture was ﬁltered, the coupling

repeated once and the resin was washed with DMF (3x2.5 mL, 0.5 min).

A mixture of DCM/MeOH/DIPEA (80/15/5, 2.5 mL) was added to the

resin, the reservoir was shaken for 10 min, the reaction mixture was

ﬁltered, the capping repeated once and the resin was washed with DMF

(3x2.5 mL, 0.5 min), MeOH (1x2.5 mL, 0.5 min) and DCM (6x2.5 mL,

0.5 min).

Fmoc removal was achieved with a solution DMF/piperidine (80:20,

4.0 mL, 20 minx2) then the resin was washed with DMF (5x2.5 mL,

8

E. Loza, et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

0.5 min), MeOH (1x2.5 mL, 0.5 min), DCM (1x2.5 mL) and DMF

(2x2.5 mL, 0.5 min) .

4.3.4. tert-Butyl (2-oxoethyl)carbamate (3f)

Compound 3f was prepared from tert-butyl (2-hydroxyethyl)carba-

mate (2f) (1.00 g, 6.2 mmol) and Dess-Martin periodinane (3.29 g,

7.8 mmol) as described for 3a. After evaporation of the solvent, the

residue was dried in vacuo to give aldehyde 3f as a light yellow oil

(0.85 g, 77%). ¹H NMR (400 MHz, CDCl₃) δ: 9.65 (s, 1H), 5.35–5.01 (m,

1H), 4.08 (d, J = 4.8 Hz, 2H), 1.45 (s, 9H). ¹H NMR spectrum was in

agreement with that reported in the literature.⁴³

Addition of next amino acids was achieved using the following con-

ditions: Fmoc-protected amino acid (3.0 equiv.), DMF (1.3 mL) then

DIPEA (4.0 equiv.) were added to the resin and the reservoir was shaken

until full dissolution. HBTU (2.9 equiv.) was added and the reservoir was

shaken for 120 min. The reaction mixture was ﬁltered, the coupling re-

peated once and the resin was washed with DMF (3x2.5 mL, 0.5 min),

MeOH (1x2.5 mL, 0.5 min) and DCM (6x2.5 mL, 0.5 min).

Cleavage from the resin and global deprotection was achieved using

the following conditions: after the last Fmoc removal the resin was

rinsed carefully with DCM (5x2.5 mL, 2 min) and dried overnight (18 h)

at room temperature. 2.0 mL of cleavage cocktail were freshly prepared

(TFA/H₂O/TIS, 85/7.5/7.5) and added to the resin. The mixture was

shaken for 3 h and the solution was added dropwise to tubes containing

30 mL of cold (0 °C) TBME. This step was repeated once, the cold

mixture was stirred at 0 °C for 30 min and the tubes were centrifuged

(2,200 g, 5 min). The supernatant was discarded, the solid was washed

with cold TBME (10 mL) and then centrifuged again (2,200 g, 5 min).

The crude product obtained was air-dried (3 h) at room temperature,

dissolved in water and freeze dried.

4.3.5. Benzyl

(3S,5S,6R)-3-methyl-2-oxo-5,6-diphenylmorpholine-4-

carboxylate ((2S)(S,R)-5)

Compound (2S)(S,R)-5 (4.51 g, 84%) was prepared from dipheny-

loxazinone (S,R)-4 (5.00 g, 12.9 mmol), methyl iodide (10.99 g,

77.4 mmol), and 2 M solution of NaHMDS in THF (9.7 mL, 19.4 mmol)

as described by Williams.^{25 1}H NMR (300 MHz, DMSO‑d₆) δ: (rotamer

mixture) 7.45–7.31 (m, 2H), 7.30–7.02 (m, 10H), 6.75–6.62 (m, 1H),

6.60–6.48 (m, 2H), 6.31(d, J = 2.7 Hz, 1H), 5.31 (d, J = 2.7 Hz, 0.6H),

2.56 (d, J = 2.7 Hz, 0.4H), 5.22–4.85 (m, 3H), 1.74 (d, J = 7.0, 3H). ¹H

NMR spectrum was in agreement to that reported in the literature.²⁵

4.3.6. Benzyl

(3R,5R,6S)-3-methyl-2-oxo-5,6-diphenylmorpholine-4-

Crude peptides were puriﬁed on semi preparative HPLC. Pure

fractions were combined, lyophilized. TFA salt was exchanged for HCl

salt by dissolving the pure peptide in aqueous HCl solution (0.05 M) at a

concentration of 20 mg/mL. The solution obtained was freeze dried and

this step was repeated twice. HCl salt of the peptide was analysed on

analytical LC-MS. All peptides were of purity > 95%.

carboxylate ((2R)(R,S)-5)

Compound (2R)(R,S)-5 (3.34 g, 64%) was prepared from dipheny-

loxazinone (R,S)-4 (5.00 g, 12.9 mmol), methyl iodide (9.16 g,

64.5 mmol), and 2 M solution of NaHMDS in THF (8.4 mL, 16.8 mmol)

as described by Williams.^{25 1}H NMR spectrum was in agreement to that

reported in the literature.²⁵

4.3. Synthesis of non-proteinogenic amino acids

4.3.7. Benzyl (3S,5S,6R)-3-((1S,2R)-2-((tert-butoxycarbonyl)amino)-1-

hydroxypropyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (6a)

A solution of diphenyloxazinone (S,R)-4 (11.00 g, 28.4 mmol) in

anhydrous DCM (300 mL) was cooled to −5 °C (dry ice/acetone bath)

under argon atmosphere and Bu₂BOTf (1 M solution in DCM, 56.8 mL,

56.8 mmol) over 15 min was added, followed by Et₃N (8.62 g,

85.2 mmol). The yellowish solution was stirred for 15 min at −5°C,

then it was cooled to −78 °C and a solution of aldehyde 3a (8.85 g,

51.1 mmol) in anhydrous DCM (40 mL) was added over 30 min. After

stirring at −78 °C for 1 h the reaction was quenched by addition of

phosphate buﬀer (pH 7) (100 mL) at − 78 °C. After warming to room

temperature layers were separated and the aqueous layer was extracted

twice with DCM. The combined organic layers were dried (Na₂SO₄),

ﬁltered, and volatiles were evaporated to aﬀord crude 6a as 85:15

mixture of diastereomers. The crude oily 6a was mixed with EtOAc

(100 mL) and hexane (300 mL), the obtained white suspension was

stirred overnight and ﬁltered. The precipitate (16 g) was suspended in

EtOAc (100 mL), stirred for 1 h, ﬁltered, and dried in vacuo to give

desired compound (3S,5S,6R)-6a (9.04 g, 56%) as a white solid (99%

de). [ ]²_D¹+ 25.9 (c 1.16, MeOH). ¹H NMR (400 MHz, DMSO‑d₆) δ: (2:1

rotamer mixture) 7.43 – 7.06 (m, 10H), 7.03–6.95 (m, 2H), 6.70 – 6.53

(m, 4H), 6.51–6.48 (m, 1H), 6.00–5.91 (m, 1H), 5.29 (d, J = 3.0 Hz,

0.67H), 5.23 (d, J = 3.0 Hz, 0.33H), 5.14 – 5.05 (m, 1.66H), 5.02 (d,

J = 13.1 Hz, 0.67H), 4.88 (d, J = 13.1 Hz, 0.67H), 4.06 – 3.97 (m, 1H),

3.85–3.68 (m, 1H), 1.39 (s, 9H), 1.28 (d, J = 6.6 Hz, 2H), 1.10 (d,

J = 6.6, 1H). ¹³C NMR (DMSO‑d₆) δ: 167.1, 167.0, 155.3, 153.8, 153.2,

136.7, 136.2, 136.1, 136.0, 134.9, 134.8, 128.3, 128.2, 128.1, 128.0,

127.7, 127.5, 127.4, 127.3, 127.2, 126.5, 126.1, 126.1, 78.3, 78.1,

77.5, 76.3, 75.4, 67.2, 66.4, 60.2, 60.0, 59.6, 48.9, 28.3, 18.0. HRMS

(ESI) m/z: Calculated for C₃₂H₃₆N₂NaO₇[M + Na]⁺583.2415, found

583.2416.

4.3.1. tert-Butyl (R)-(1-oxopropan-2-yl)carbamate (3a)

To a solution of tert-butyl (R)-(1-hydroxypropan-2-yl)carbamate

(2a) (10.00 g, 57.1 mmol) in DCM (300 mL) was added solid Dess-

Martin periodinane (33.89 g, 79.9 mmol) in one portion. Water

(1.4 mL) was added dropwise within 0.5 h to the vigourously stirred

suspension, and the stirring was continued for 2 h at room temperature,

whereupon EtOAc (1000 mL) was added. The suspension was ﬁltered

through a pad of Celite, and the ﬁlter-cake was washed with EtOAc

(500 mL). Combined ﬁltrates were washed sequentially with aqueous

saturated NaHCO₃, aqueous 10% Na₂S₂O₃(1:1), brine, and dried

(Na₂SO₄). Volatiles were evaporated and the residue was dried in vacuo

to give aldehyde 3a as a white solid (9.30 g, 94%). ¹H NMR (400 MHz,

CDCl₃) δ: 9.56 (s, 1H), 5.09 (m, 1H), 4.23 (qui, J = 7.3 Hz, 1H), 1.45 (s,

9H), 1.33 (d, J = 7.3 Hz, 3H). ¹H NMR spectrum was in agreement with

that reported in the literature.⁴⁰

4.3.2. tert-Butyl methyl(2-oxoethyl)carbamate (3d)

Compound 3d was prepared from tert-butyl (2-hydroxyethyl)(me-

thyl)carbamate (2d) (1.00 g, 5.7 mmol) and Dess-Martin periodinane

(3.03 g, 7.1 mmol) as described for 3a. After evaporation of the solvent,

the residue was dried in vacuo to give aldehyde 3d as a colorless oil

(0.77 g, 78%). ¹H NMR (400 MHz, CDCl₃) δ: (1:1 rotamer mixture) 9.60

(s, 1H), 4.02 (s, 1H), 3.90 (s, 1H), 3.00–2.88 (m, 3H), 1.51–1.39 (m,

9H). ¹H NMR spectrum was in agreement with that reported in the

literature.⁴¹

4.3.3. tert-Butyl (3-oxopropyl)carbamate (3e)

Compound 3e was prepared from tert-butyl (3-hydroxypropyl)car-

bamate (2e) (10.87 g, 62.0 mmol) and Dess-Martin periodinane

(31.57 g, 74.4 mmol) as described for 3a. After evaporation of the

solvent, the residue was puriﬁed by column chromatography on silica

gel (eluent petroleum ether/EtOAc, 1:1) to provide aldehyde 3e as a

colorless oil (8.50 g, 79%). ¹H NMR (400 MHz, CDCl₃) δ: 9.81 (t,

J = 0.9 Hz, 1H), 4.88 (br s, 1H), 3.42 (q, J = 6.0 Hz, 2H), 2.70 (t,

J = 5.9 Hz, 2H), 1.43 (s, 9H). ¹H NMR spectrum was in agreement with

that reported in the literature.⁴²

4.3.8. Benzyl (3S,5S,6R)-3-((1S,2S)-2-((tert-butoxycarbonyl)amino)-1-

hydroxypropyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (6b)

Compound 6b was prepared from diphenyloxazinone (S,R)-4

(4.70 g, 12.1 mmol), Bu₂BOTf (1 M solution in DCM, 20.2 mL,

20.2 mmol), Et₃N (4.2 mL, 30.3 mmol), and tert-butyl (S)-(1-oxopropan-

2-yl)carbamate (3b) (1.75 g, 10.1 mmol) as described for 6a. After

9

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Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

evaporation of the solvent, the oily residue was puriﬁed by column

chromatography on silica gel (eluent petroleum ether/EtOAc, gradient

from 5:1 to 1:1) followed by crystallization from EtOAc to give the title

compound (3S,5S,6R)-6b (1.60 g, 28%) as a white solid (99% de).

[ ]²_D¹+ 42.0 (c 1.0, CHCl₃). ¹H NMR (400 MHz, DMSO‑d₆) δ: (rotamer

mixture, 2:1) 7.35 – 7.04 (m, 10H), 7.03–6.97 (m, 2H), 6.70 – 6.34 (m,

5H), 6.19 (d, J = 5.7 Hz, 0.33H), 6.17 (d, J = 5.7 Hz, 0.67H), 5.28 (d,

J = 3.0 Hz, 0.67H), 5.22 (d, J = 3.0 Hz, 0.33H), 5.19 (d, J = 12.7 Hz,

0.33H), 5.11 (d, J = 12.7 Hz, 0.33H), 5.07 (s, 0.67H), 5.04 (s, 0.33H),

5.01 (d, J = 13.1 Hz, 0.67H), 4.88 (d, J = 13.1 Hz, 0.67H), 4.03–3.80

(m, 2H), 1.41 (s, 6H), 1.36 (s, 3H), 1.14 (d, J = 6.5 Hz, 2H), 1.09 (d,

J = 6.5 Hz, 1H). HRMS (ESI) m/z: Calculated for C₃₂H₃₆N₂NaO₇

[M + Na]⁺583.2415, found 583.2401.

carbamate (3e) (8.05 g, 46.5 mmol) as described for 6a. After eva-

poration of the solvent, the oily residue of crude 6e (88:12 d.r.) was

puriﬁed by column chromatography on silica gel (eluent petroleum

ether/DCM/EtOAc, 1:3:1) followed by crystallization from EtOAc/

hexane to give the desired (3S,5S,6R)-6e (5.40 g, 37%) as a white solid.

[ ]²_D¹−14 (c 1.0, MeOH). ¹H NMR (400 MHz, DMSO‑d₆) δ: (rotamer

mixture, 7:3) 7.42–7.06 (m, 10H), 7.02–6.95 (m, 2H), 6.82 (t,

J = 5.5 Hz, 0.7H), 6.78 (t, J = 5.5 Hz, 0.3H), 6.73–6.67 (m, 1H),

6.59–6.52 (m, 2H), 6.48 (d, J = 3.0 Hz, 0.7H), 6.46 (d, J = 3.0 Hz,

0.3H), 5.98 (d, J = 5.3 Hz, 0.3H), 5.95 (d, J = 5.3 Hz, 0.7H), 5.27 (d,

J = 3.0 Hz, 0.7H), 5.22 (d, J = 3.0 Hz, 0.3H), 5.14 (d, J = 12.5 Hz,

0.3H), 5.05 (d, J = 12.5 Hz, 0.3H), 5.02 (d, J = 13.0 Hz, 0.7H), 4.90

(d, J = 13.0 Hz, 0.7H), 4.87–4.85 (m, 0.3H), 4.81–4.79 (m, 0.7H),

4.21–4.11 (m, 1H), 3.19–2.96 (m, 2H), 1.89–1.78 (m, 1H), 1.78–1.66

(m, 1H), 1.40 (s, 6.3H), 1.39 (s, 2.7H). ¹³C NMR (101 MHz, DMSO‑d₆)

δ: 167.0, 166.9, 155.7, 153.8, 153.1, 136.6, 136.1, 136.0, 134.9, 134.8,

128.5, 128.2, 128.0, 127.8, 127.6, 127.5, 127.3, 127.2, 126.6, 126.1,

126.1, 78.4, 78.2, 77.5, 71.5, 70.2, 67.0, 66.5, 62.8, 62.7, 59.5, 59.5,

40.2, 37.2, 34.4, 28.3, 28.3. HRMS (ESI) m/z: Calculated for

4.3.9. Benzyl (3S,5S,6R)-3-((R)-2-((tert-butoxycarbonyl)amino)-1-hydroxy-

2-methylpropyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (6c)

Compound 6c was prepared from diphenyloxazinone (S,R)-4

(5.20 g, 13.4 mmol), Bu₂BOTf (1 M solution in DCM, 26.9 mL,

26.9 mmol), Et₃N (5.6 mL, 40.2 mmol), and tert-butyl (2-methyl-1-ox-

opropan-2-yl)carbamate (3c) (4.52 g, 24.2 mmol) as described for 6a.

After evaporation of the solvent, the oily residue of crude 6c (83:17

d.r.) was puriﬁed by crystallization from EtOAc/hexane to give

(3S,5S,6R)-6c (6.89 g, 52%) as a white solid. [ ]²_D¹−18.7 (c 1.1,

MeOH). ¹H NMR (DMSO‑d₆) δ: (rotamer mixture, 4:1) 7.44 – 7.04 (m,

10H), 7.04–6.95 (m, 2H), 6.74–6.49 (m, 5H), 6.47 (d, J = 6.3 Hz,

0.2H), 6.42 (d, J = 6.4 Hz, 0.8H), 5.30 (d, J = 3.1 Hz, 0.8H), 5.24 (d,

J = 3.1 Hz, 0.2H), 5.22 (s, 0.2H), 5.19 (s, 0.8H), 5.13 (d, J = 12.5 Hz,

0.2H), 5.09 (d, J = 12.5 Hz, 0.2H), 4.96 (d, J = 12.9 Hz, 0.8H), 4.85

(d, J = 12.9 Hz, 0.8H), 4.27 (d, J = 5.9 Hz, 0.2H), 4.13 (d, J = 6.4 Hz,

0.8H), 1.43 (s, 2.4H), 1.39 (s, 7.2H), 1.39 (s, 1.8H), 1.37 (s, 0.6H), 1.25

(s, 0.6H). ¹³C NMR (DMSO‑d₆) δ: 167.4, 154.9, 153.9, 136.8, 135.9,

134.7, 128.3, 128.2, 128.0, 127.7, 127.5, 127.4, 127.1, 126.7, 126.1,

79.3, 77.8, 66.7, 60.2, 59.7, 56.0, 28.3, 22.5. HRMS (ESI) m/z:

Calculated for C₃₃H₃₈N₂NaO₇[M + Na]⁺597.2571, found 597.2593.

C

₃₂H₃₆N₂NaO₇[M + Na]⁺583.2415, found 583.2418.

4.3.12. Benzyl

(3S,5S,6R)-3-((S)-2-((tert-butoxycarbonyl)amino)-1-

hydroxyethyl)-3-methyl-2-oxo-5,6-diphenylmorpholine-4-carboxylate (6f)

Benzyl (2R,3S)-6-((tert-butyldimethylsilyl)oxy)-5-methyl-2,3-diphenyl-

2,3-dihydro-4H-1,4-oxazine-4-carboxylate. To a solution of compound

(2S)(S,R)-5 (13.17 g, 32.8 mmol) and tert-butyldimethylsilyl chloride

(5.29 g, 35.1 mmol) in THF (200 mL) at –78 °C was added NaHMDS

(2 M solution in THF, 17.2 mL, 34.4 mmol) in THF and the resulting

mixture was stirred at the same temperature for 1 h. The mixture was

warmed to room temperature and water (150 mL) was added. The

obtained mixture was extracted with Et₂O (150 mL), the organic layer

was washed with brine, and dried (Na₂SO₄)_.The solvent was evapo-

rated and the residue (20 g) was puriﬁed by reverse phase column

chromatography (gradient elution form 70% MeCN in water to 100%

MeCN) to give benzyl (2R,3S)-6-((tert-butyldimethylsilyl)oxy)-5-me-

thyl-2,3-diphenyl-2,3-dihydro-4H-1,4-oxazine-4-carboxylate as a white

solid (9.66 g, 57%). ¹H NMR spectrum (300 MHz, DMSO‑d₆) δ:

7.40–7.21 (m, 8H), 7.18–7.06 (m, 5H), 6.80–6.72 (m, 2H), 5.52 (d,

J = 3.1 Hz, 1H), 5.47 (d, J = 3.1 Hz, 1H), 5.16 (d, J = 14.5 Hz, 1H),

5.12 (d, J = 14.5 Hz, 1H), 2.03 (s, 3H), 0.93 (s, 9H), 0.16 (s, 3H), 0.15

(s, 3H).

4.3.10. Benzyl (3S,5S,6R)-3-((S)-2-((tert-butoxycarbonyl)(methyl)amino)-

1-hydroxyethyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (6d)

Compound 6d was prepared from diphenyloxazinone (S,R)-4

(3.50 g, 9.0 mmol), Bu₂BOTf (1 M solution in DCM, 18.1 mL,

18.1 mmol), Et₃N (3.8 mL, 27.3 mmol), and tert-butyl methyl(2-ox-

oethyl)carbamate (3d) (3.00 g, 17.3 mmol) as described for 6a. After

evaporation of the solvent, the oily residue of crude 6d (4:1 d.r.) was

puriﬁed by reversed-phase column chromatography (gradient elution

from 50% MeCN in water to 100% MeCN) to give a white solid (3.98 g),

which was further puriﬁed by multiple direct-phase column chroma-

tography on silica gel (eluent petroleum ether/DCM/EtOAc, 9:9:2) to

give the title compound (3S,5S,6R)-6d (1.76 g, 35%). Additionally, a

fraction containing less pure 6d (77:23 d.r., 0.88 g) was also obtained.

[ ]²_D²+ 49.4 (c 1.0, CH₂Cl₂). ¹H NMR (400 MHz, DMSO‑d₆) δ: (rotamer

mixture) 7.47–7.05 (m, 9.75H), 7.04–6.93 (m, 2H), 6.78–6.66 (m,

1.25H), 6.62–6.51 (m, 2H), 6.48–6.38 (m, 1H), 6.27–6.13 (m, 1H),

5.33–5.18 (m, 1H), 5.15–4.83 (m, 3H), 4.43–4.33 (m, 0.85H),

4.32–4.22 (m, 0.15H), 3.63–3.50 (m, 0.5H), 3.46–3.37 (m, 1H),

3.30–3.21 (m, 0.5H), 2.91 (s, 2H), 2.81 (s, 0.5H), 2.70 (s, 0.5H), 1.43 (s,

4.5H), 1.41 (s, 2.25H), 1.36 (s, 2.25H). ¹³C NMR (101 MHz, DMSO‑d₆)

δ: (rotamer mixture) 167.1, 154.8, 153.7, 136.6, 136.3, 136.0, 134.8,

134.7, 128.3, 128.0, 128.0, 127.8, 127.8, 127.5, 127.2, 126.7, 126.1,

78.5, 78.4, 71.7, 71.3, 66.5, 60.6, 59.4, 52.0, 51.6, 35.7, 28.1. HRMS

(ESI) m/z: Calculated for C₃₂H₃₇N₂O₇[M + H]⁺561.2595, found

561.2604.

To a solution of above prepared benzyl (2R,3S)-6-((tert-butyldi-

methylsilyl)oxy)-5-methyl-2,3-diphenyl-2,3-dihydro-4H-1,4-oxazine-4-

carboxylate (6.74 g, 13.1 mmol) and aldehyde 3f (3.12 g, 19.6 mmol)

in THF (120 mL) was added tetrabutylammonium ﬂuoride (1 M solu-

tion in THF, 26.2 mL, 26.2 mmol) at −78 °C and the resulting mixture

was stirred at −78 °C for 1 h . The reaction mixture was quenched with

cold water (300 mL), supplemented with EtOAc (300 mL), and the

mixture was allowed to warm to room temperature. The organic layer

was separated, washed successively with water (4 × 300 mL), brine,

and dried (Na₂SO₄). After evaporation of the solvent, the crude 6f

(83:17 d.r.) was puriﬁed by reversed-phase column chromatography

(gradient elution from 70% MeCN in water 100% MeCN), followed by

column chromatography on silica gel (gradient elution from 5:1 pet-

roleum ether/EtOAc to 3:1 petroleum ether/EtOAc) to give the desired

(3S,5S,6R)-6f as a white solid (2.98 g, 38%). [ ]²_D²+ 30.0 (c 1.0,

DCM). ¹H NMR spectrum (300 MHz, DMSO‑d₆) δ: (rotamer mixture,

3:1) 7.47–6.66 (m, 16H), 6.57–6.45 (m, 1H), 6.36–6.19 (m, 1H),

5.53–5.32 (m, 1H), 5.23–4.94 (m, 2H), 4.82–4.50 (m, 1H), 3.46–3.34

(m, 1H, overlapped with water), 2.87–2.54 (m, 1H), 1.86 (br s, 2.25H),

1.77 (br s, 0.75H), 1.47–1.30 (m, 9H). ¹³C NMR (101 MHz, DMSO‑d₆) δ:

170.2, 155.7, 152.9, 136.5, 136.2, 135.2, 128.2, 128.1, 128.0, 127.9,

127.7, 127.6, 127.1, 125.8, 78.4, 77.7, 71.8, 67.4, 66.5, 60.4, 42.8,

28.3, 22.0. HRMS (ESI) m/z: Calculated for C₃₂H₃₇N₂O₇[M + H]⁺

561.2595. Found: 561.2599.

4.3.11. Benzyl

(3S,5S,6R)-3-((S)-3-((tert-butoxycarbonyl)amino)-1-

hydroxypropyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (6e)

Compound 6e was prepared from diphenyloxazinone (S,R)-4

(10.00 g, 25.8 mmol), Bu₂BOTf (1 M solution in DCM, 51.6 mL,

51.6 mmol), Et₃N (10.8 mL, 77.5 mmol), and tert-butyl (3-oxopropyl)

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4.3.13. Benzyl

(3S,5S,6R)-3-((R)-2-((tert-butoxycarbonyl)amino)-1-

54.5, 28.4, 24.0, 23.4. HRMS (ESI) m/z: Calculated for C₁₁H₂₂N₂NaO₅

hydroxyethyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (6 g)

[M + Na]⁺285.1421, found 285.1428.

To a solution of diphenyloxazinone (S,R)-4 (11.62 g, 30.0 mmol) in

dry THF (650 mL) under Ar atmosphere at − 78 °C were added

NaHMDS (2 M solution in THF, 15 mL, 30.0 mmol) followed by cold

solutions of anhydrous ZnBr₂(6.76 g, 30.0 mmol) in anhydrous THF

(25 mL) and tert-butyl (2-oxoethyl)carbamate (3f) (2.388 g, 15.0 mmol)

in anhydrous THF (15 mL) and the resulting mixture was stirred

at − 78 °C for 1 h. The reaction mixture was allowed to warm to room

temperature, stirred at this temperature for 1 h, and quenched with

saturated aqueous NH₄Cl solution (150 mL). The organic layer was

separated and the aqueous layer was extracted with DCM

(2 × 100 mL). The organic layers were combined, washed with brine

(100 mL), and dried (Na₂SO₄). Solvents were evaporated and the light

yellow solid residue of crude 6 g (83:17 d.r., 15.8 g) was puriﬁed by

silica gel chromatography (gradient elution from 15:5:1 petroleum

ether/EtOAc/THF to to 13:7:1 petroleum ether/EtOAc/THF) to give the

desired (3S,5S,6R)-6g (2.17 g, 26%) as a white solid. [ ]²_D²−20.0 (c 1.1,

DCM). ¹H NMR (400 MHz, DMSO‑d₆) δ: 7.43–7.29 (m, 1H), 7.28–7.03

(m, 9H), 7.02–6.94 (m, 2H), 6.68–6.46 (m, 5H), 6.10 (d, J = 6.7 Hz,

1H), 5.28 (d, J = 2.8 Hz, 1H), 5.12–4.83 (m, 3H), 4.16–3.99 (m, 1H),

3.29–2.90 (m, 2H), 1.41 (s, 9H). ¹³C NMR (101 MHz, DMSO‑d₆) δ:

169.1, 155.6, 155.5, 153.9, 136.8, 136.4, 136.0, 135.7, 134.7, 128.3,

128.1, 128.0, 128.0, 127.8, 127.8, 127.5, 127.4, 127.3, 127.2, 126.9,

126.8, 126.1, 78.0, 77.7, 72.6, 70.9, 67.0, 60.2, 59.9, 44.0, 28.2, 28.1.

HRMS (ESI) m/z: Calculated for C₃₁H₃₅N₂O₇[M + H]⁺547.2439.

Found: 547.2444.

4.3.17. (2S,3S)-2-Amino-4-((tert-butoxycarbonyl)(methyl)amino)-3-

hydroxybutanoic acid (7d)

Compound 7d was prepared from 6d (1.76 g, 3.1 mmol) and 10%

Pd on activated carbon (0.84 g, 0.8 mmol) as described for 7a. After

evaporation, the residue was triturated with Et₂O, ﬁltered, and dried in

vacuo to give the crude product 7d (0.77 g, 98%) as a white solid, which

was used in the next step without additional puriﬁcation. ¹H NMR

(400 MHz, D₂O) δ: 4.26–3.91 (m, 1H), 3.61–3.17 (m, 3H), 2.91 (s, 3H),

1.46 (s, 9H). LCMS (ESI) m/z: 249.4 [M + H]⁺

.

4.3.18. (2S,3S)-2-Amino-5-((tert-butoxycarbonyl)amino)-3-

hydroxypentanoic acid (7e)

Compound 7e was prepared from 6e (5.39 g, 9.6 mmol) and 10% Pd

on activated carbon (2.05 g, 1.9 mmol) as described for 7a. Product 7e

(white solid, 2.30 g, 96%) was obtained suﬃciently pure to be used

directly in the next step. [ ]²_D¹–26 (c 1.0, MeOH). ¹H NMR (400 MHz,

CD₃OD) δ: 4.12 (dt, J = 8.2, 4.3, 1H), 3.67 (d, J = 4.0, 1H), 3.26–3.12

(m, 2H), 1.72 – 1.57 (m, 2H), 1.43 (s, 9H). ¹³C NMR (101 MHz, CD₃OD)

δ: 171.8, 158.7, 80.0, 68.2, 61.2, 38.3, 32.4, 28.8. HRMS (ESI) m/z:

Calculated for C₁₀H₂₀N₂NaO₅(M + Na)⁺271.1264, found 271.1275.

4.3.19. (2S,3S)-2-Amino-4-((tert-butoxycarbonyl)amino)-3-hydroxy-2-

methylbutanoic acid (7f)

Hydrogen gas was passed through a mixture of compound 6f

(3.00 g, 5.4 mmol) and 10% Pd on activated carbon (1.42 g, 1.4 mmol)

in MeOH (100 mL) at room temperature for 1 h. The black suspension

was ﬁltered through a pad of silica gel and washed with MeOH (20 mL).

The ﬁltrate was evaporated and the residue (2.4 g) was triturated with

MeCN and Et₂O to provide 7f (1.30 g, 70% purity), which was used in

the next step without additional puriﬁcation. LCMS (ESI) m/z: 249.4

[M + H]⁺. ¹H NMR (300 MHz, D₂O) δ: 3.72 (dd, J = 9.7, 2.8 Hz, 1H),

3.26 (d, J = 13.5 Hz, 1H), 3.07 (dd, J = 13.5, 9.7 Hz, 1H), 1.45 (s, 9H),

1.29 (s, 3H).

4.3.14. (2S,3R,4R)-2-Amino-4-((tert-butoxycarbonyl)amino)-3-

hydroxypentanoic acid (7a)

Hydrogen gas was passed through a mixture of compound 6a

(9.00 g, 16.1 mmol) and 10% Pd on activated carbon (3.42 g,

3.2 mmol) in 1:1 (v:v) mixture of MeOH and THF (30 mL) for 2 h at

room temperature. The black suspension was ﬁltered through a short

column of silica gel and the column was washed with MeOH (100 mL)

and 1:1 (v:v) mixture of i–PrOH and H₂O (60 mL). Combined ﬁltrates

were evaporated, the residue was triturated with MeCN (20 mL), ﬁl-

tered, and dried in vacuo to give 7a as a white solid (1.75 g, 44%),

which was used in the next step without further puriﬁcation. [ ]²_D¹+7.0

(c 1.0, H₂O-CF₃COOH, 130:1). ¹H NMR (400 MHz, DMSO‑d₆) δ: 7.54

(br s, 4H), 6.81 (d, J = 8.2 Hz, 1H), 3.66–3.58 (m, 1H), 3.49 (dd,

J = 9.1, 2.2 Hz, 1H), 2.87 (d, J = 9.1 Hz, 1H), 1.39 (s, 9H), 1.10 (d,

J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆) δ: 169.0, 168.7, 155.7,

77.9, 72.7, 60.1, 54.7, 48.4, 28.3, 22.4, 17.8. HRMS (ESI) m/z:

Calculated for C₁₀H₂₀N₂NaO₅[M + Na]⁺271.1264, found 271.1277.

4.3.20. (2S,3R)-2-Amino-4-((tert-butoxycarbonyl)amino)-3-

hydroxybutanoic acid (7 g)

Hydrogen gas was passed through a mixture of compound 6 g

(1.90 g, 3.5 mmol) and 10% Pd on activated carbon (0.74 g, 0.7 mmol)

in 2:1 (v:v) mixture of MeOH and THF (120 mL) for 1 h at room tem-

perature. The reaction mixture was ﬁltered through a pad of silica gel

and washed with MeOH (200 mL). The ﬁltrate was evaporated and the

residue (1.38 g) was triturated with MeCN and Et₂O to provide the

crude 7 g (0.67 g, 82%), which was used in the next step without

further puriﬁcation. ¹H NMR (400 MHz, D₂O) δ: 4.21–4.00 (m, 1H),

3.70–3.51 (m, 1H), 3.46–3.26 (m, 1H), 3.26–3.06 (m, 1H), 1.45 (s, 9H).

4.3.15. (2S,3R,4S)-2-Amino-4-((tert-butoxycarbonyl)amino)-3-

hydroxypentanoic acid (7b)

Compound 7b was prepared from 6b (1.00 g, 1.8 mmol) and 10%

Pd on activated carbon (0.38 g, 0.4 mmol) as described for 7a. Product

7b was obtained as a white solid (0.40 g, 90%), which was suﬃciently

pure to be used directly in the next step. [ ]²_D¹+ 7.8 (c 1.0, MeOH).

HRMS (ESI) m/z: Calculated for C₁₀H₁₉N₂O₅[M−H]^-247.1299, found

247.1300. Since a quality of recorded ¹H NMR and ¹³C NMR spectra

was insuﬃcient for reliable characterization, the complete character-

ization of 7b was performed for the downstream Fmoc-derivative 8b.

LCMS ESI (m/z): 235.6 [M + H]⁺

.

4.3.21. (2S,3S,4R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-

((tert-butoxycarbonyl)amino)-3-hydroxypentanoic acid (8a)

To a solution of compound 7a (1.73 g, 7.0 mmol) and NaHCO₃

(1.76 g, 20.9 mmol) in water (200 mL) was added dioxane (50 mL) and

the mixture was cooled in an ice bath. To the mixture a solution of

FmocOSu (2.35 g, 7.0 mmol) in dioxane (50 mL) was added drop-wise

over 15 min, then the reaction mixture was allowed to warm to room

temperature and stirred overnight. The mixture was supplemented with

water (50 mL) and washed with Et₂O (3 × 40 mL). EtOAc (80 mL) was

added to the aqueous layer and the mixture was acidiﬁed with 6 M HCl

to pH 3. The organic layer was separated and washed successively with

1 M aqueous solution of KHSO₄(30 mL), water (30 mL), brine (30 mL),

and dried (Na₂SO₄). The solvents were evaporated and the white solid

residue was crystallized from MeCN (20 mL) to give (2S,3S,4R)-8a as a

white solid (2.31 g, 70%). [ ]²_D¹−2.0 (c 1.1, MeOH). ¹H NMR

(400 MHz, DMSO‑d₆) δ: 12.35 (br s, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74

4.3.16. (2S,3R)-2-Amino-4-((tert-butoxycarbonyl)amino)-3-hydroxy-4-

methylpentanoic acid (7c)

Compound 7c was prepared from 6c (6.80 g, 11.3 mmol) and 10%

Pd on activated carbon (1.46 g, 1.4 mmol) as described for 7a. Product

7c was obtained as a white solid (1.60 g, 52%), which was suﬃciently

pure to be used directly in the next step. [ ]²_D¹–6.8 (c 0.62, H₂O/MeOH,

3:2). ¹H NMR (400 MHz, DMSO‑d₆+ TFA) δ: 8.24 (br d, J ~ 5.0 Hz,

3H), 6.44 (s, 1H), 3.97–3.91 (m, 2H), 1.37 (s, 9H), 1.28 (s, 3H), 1.25 (s,

3H). ¹³C NMR (101 MHz, DMSO‑d₆) δ: 168.5, 154.8, 76.8, 76.6, 55.0,

11

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Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

(d, J = 7.4 Hz, 1H), 7.68 (d, J = 7.4 Hz, 1H), 7.57 (d, J = 9.1 Hz, 1H),

7.42 (t, J = 7.5 Hz, 2H), 7.36–7.30 (m, 2H), 6.17 (d, J = 9.0 Hz, 1H),

4.32 (dd, J = 9.2, 6.8 Hz, 1H), 4.23 (t, J = 6.7 Hz, 1H), 4.14 (t,

J = 8.6 Hz, 1H), 4.00 (t, J = 8.4 Hz, 1H), 3.86–3.71 (m, 1H), 3.59 (dd,

J = 8.1, 3.2 Hz, 1H), 1.35 (s, 9H), 1.05 (d, J = 6.7 Hz, 3H). ¹³C NMR

(101 MHz, DMSO‑d₆) δ: 172.9, 155.6, 155.0, 144.1, 143.6, 140.7,

140.6, 127.6, 127.1, 127.1, 125.5, 125.1, 120.1, 77.6, 73.0, 65.8, 56.6,

46.6, 46.5, 28.2, 18.4. HRMS (ESI) m/z: Calculated for C₂₅H₃₀N₂NaO₇

1.41 and 1.36 (s and s, altogether 9H). ¹³C NMR (101 MHz, DMSO‑d₆)

δ: (rotamer mixture) 171.6, 156.1, 155.4, 154.8, 143.8, 140.7, 127.6,

127.1, 125.3, 120.1, 78.9, 78.5, 70.2, 68.9, 65.8, 64.9, 57.9, 51.5, 50.7,

46.6, 35.4, 28.1, 15.2. HRMS (ESI) m/z: Calculated for C₂₅H₃₁N₂O₇

[M

+

H]⁺471.2126, found 471.2117. Anal. Calcd for

C₂₅H₃₀N₂O₇× 0.44 H₂O (1.6%) × 0.23 EtOH (2.2%): 62.53, H 6.65, N

5.73. Found: C 62.52, H 6.53, N 5.57. The presence of EtOH (ca.

0.23 eq, 2.2%) was detected by ¹H NMR. Even by continuous drying in

vacuo at room temperature we were not able to remove this impurity

completely.

[M

+

Na]⁺493.1945, found 493.1933. Anal. Calcd for

C

₂₅H₃₀N₂O₇× 0.11 H₂O (0.4%) × 0.04 MeCN (0.3%): C 63.53, H 6.45,

N 6.03. Found: C 63.54, H 6.38, N 6.03. The presence of MeCN (ca.

0.04 eq, 0.3%) was detected by ¹H NMR. Traces of MeCN could not be

removed after prolonged drying in vacuo at room temperature.

4.3.25. (2S,3S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-

((tert-butoxycarbonyl)amino)-3-hydroxypentanoic acid (8e)

Compound 8e was prepared from 7e (2.30 g, 9.3 mmol), solid

NaHCO₃(1.95 g, 23.2 mmol), and FmocOSu (3.12 g, 9.3 mmol) as

described for 8a. Crystallization from MeCN furnished (2S,3S)-8e as

white crystals (3.10 g, 71%). Mp: 123–124 °C (dec.). [ ]²_D¹+1.7 (c 1.0,

MeOH). ¹H NMR (400 MHz, DMSO‑d₆) δ: 12.54 (br s, 1H), 7.84 (d,

J = 7.6 Hz, 2H), 7.73 (d, J = 7.7 Hz, 2H), 7.46 (d, J = 8.5 Hz, 1H),

7.42 (td, J = 7.5, 0.9 Hz, 2H), 7.33 (td, J = 7.4, 1.1 Hz, 2H), 6.71 (t,

J = 5.4 Hz, 1H), 4.98 (br s, 1H), 4.30–4.24 (m, 2H), 4.22 (t, J = 6.8 Hz,

1H), 3.97 (dd, J = 8.5, 5.8 Hz, 1H), 3.79–3.71 (m, 1H), 3.12–3.01 (m,

1H), 3.01–2.91 (m, 1H), 1.65–1.49 (m, 2H), 1.37 (s, 9H). ¹³C NMR

(101 MHz, DMSO‑d₆) δ: 172.1, 156.0, 155.6, 143.8, 140.7, 127.6,

127.1, 125.3, 120.1, 77.4, 68.4, 65.8, 59.5, 46.6, 37.1, 33.2, 28.3.

HRMS (ESI) m/z: Calculated for C₂₅H₃₀N₂NaO₇(M + Na)⁺493.1945,

found 493.1948. Anal. Calcd for C₂₅H₃₀N₂O₇× 0.11 H₂O (0.4%): C

63.55, H 6.45, N 5.93. Found: C 63.56, H 6.45, N 5.95.

4.3.22. (2S,3S,4S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-

((tert-butoxycarbonyl)amino)-3-hydroxypentanoic acid (8b)

Compound 8b was prepared from 7b (1.06 g, 4.3 mmol), solid

NaHCO₃(0.90 g, 10.7 mmol), and FmocOSu (1.44 g, 4.7 mmol) as

described for 8a. After crystallization from MeCN, the product 8b was

obtained as a white solid (1.02 g, 51%). [ ]²_D¹+4.8 (c 1.0, MeOH). ¹H

NMR (400 MHz, DMSO‑d₆) δ: (rotamer mixture, ca 9:1) 12.51 (br s,

1H), 7.89 (d, J = 7.5 Hz, 2H), 7.77–7.70 and 7.70–7.62 (m and m, 1.8H

and 0.2H), 7.44 (d, J = 9.7 Hz, 1H), 7.42 (td, J = 7.5, 1.0 Hz, 2H), 7.33

(td, J = 7.4, 1.0 Hz, 1H), 7.31 (td, J = 7.4, 1.0 Hz, 1H), 6.97–6.88 and

6.62 (m and d, J = 8.7 Hz, 0.1H and 0.9H), 5.17 (br s, 1H), 4.39–4.16

(m, 3H), 4.15 (dd, J = 9.7, 4.9 Hz, 1H), 3.73 (dd, J = 6.9, 4.9 Hz, 1H),

3.63 (dqui, J = 8.7, 6.9 Hz, 1H), 1.41 and 1.37 (s and s, 8.1H and

0.9H), 1.04 (d, J = 6.6 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆) δ:

171.8, 156.1, 155.4, 143.8, 143.7, 140.7, 127.6, 127.1, 125.3, 120.1,

78.1, 73.8, 65.9, 57.4, 47.2, 46.6, 28.2, 16.5. HRMS (ESI) m/z:

Calculated for C₂₅H₃₀N₂NaO₇[M + Na]⁺493.1945, found 493.1940.

4.3.26. (2S,3S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-

((tert-butoxycarbonyl)amino)-3-hydroxy-2-methylbutanoic acid (8f)

To a solution of crude compound 7f (1.30 g, LCMS purity ~ 70%,

3.8 mmol) in water (60 mL) at ice-bath temperature was added solid

NaHCO₃(1.10 g, 13.1 mmol) followed by slow addition (within 15 min)

of a solution of FmocOSu (1.94 g, 5.8 mmol) in dioxane (30 mL). The

obtained mixture was stirred at room temperature for 18 h, then cooled

down in an ice bath and the mixture was supplemented with solid

NaHCO₃(0.66 g, 7.9 mmol) and a solution of FmocCl (1.36 g,

5.2 mmol) in dioxane (30 mL). After stirring at room temperature for

24 h, the mixture was washed with Et₂O (2 × 150 mL) and acidiﬁed to

4.3.23. (2S,3R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-

((tert-butoxycarbonyl)amino)-3-hydroxy-4-methylpentanoic acid (8c)

Compound 8c was prepared from 7c (2.34 g, 8.9 mmol), solid

NaHCO₃(1.87 g, 22.3 mmol), and FmocOSu (3.01 g, 8.9 mmol) as

described for 8a. After evaporation of the solvent, the obtained color-

less oil was puriﬁed by reverse phase column chromatography (gradient

elution form 20% MeCN in water to 90% MeCN in water) to give

(2S,3R)-8c as a white solid (2.09 g, 48%). [ ]²_D¹−5.8 (c 1.0, MeOH). ¹H

NMR (400 MHz, DMSO‑d₆) δ: 12.32 (br s, 1H), 7.89 (d, J = 7.5 Hz, 2H),

7.71 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 8.6 Hz, 1H), 7.42 (t, J = 7.5 Hz,

2H), 7.32 (td, J = 7.5, 1.0 Hz, 1H), 7.31 (td, J = 7.5, 1.0 Hz, 1H), 6.35

(br s, 1H), 5.61 (br s, 1H), 4.29–4.17 (m, 4H), 3.77 (d, J = 6.0 Hz, 1H),

1.36 (s, 9H), 1.23 (s, 3H), 1.18 (s, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

δ: 172.3, 155.4, 155.2, 143.7, 143.7, 140.7, 127.6, 127.1, 125.3, 120.1,

77.9, 75.3, 65.7, 56.7, 55.7, 46.6, 28.2, 23.8, 23.3. HRMS (ESI) m/z:

Calculated for C₂₆H₃₂N₂NaO₇[M + Na]⁺507.2102, found 507.2102.

Anal. Calcd for C₂₆H₃₂N₂O₇× 0.06 H₂O (0.2%) × 0.12 MeCN (1.0%):

C 64.25, H 6.67, N 6.05. Found: C 64.24, H 6.65, N 6.05. The presence

of MeCN (ca. 0.12 eq, 1.0%) was detected by ¹H NMR. Traces of MeCN

could not be removed after prolonged drying in vacuo at room tem-

perature.

pH

5 with 6 M HCl. The mixture was extracted with EtOAc

(2 × 150 mL), the organic extracts combined, washed with brine, and

dried (Na₂SO₄). After evaporation of the solvent, the obtained residue

(1.7 g) was puriﬁed by reverse phase column chromatography (gradient

elution from 40% MeCN in water to 100% MeCN) to give (2S,3S)-8f as a

white solid (0.60 g, 23% over 2 steps). [ ]²_D²−15.7 (c 1.0, MeOH). ¹H

NMR (400 MHz, DMSO‑d₆) δ: (rotamer mixture, 85:15) 7.89 (d,

J = 7.5 Hz, 2H), 7.72(d, J = 7.5 Hz, 2H), 7.41 (td, J = 7.5, 0.8 Hz,

2H), 7.33 (td, J = 7.5, 0.8 Hz, 1H), 7.33 (td, J = 7.5, 0.8 Hz, 1H), 7.15

(br s, 0.85H), 6.99 (br s, 0.15H), 6.59 (t, J = 5.5 Hz, 0.85H),

6.21–6.11(m, 0.15H), 4.30–4.18 (m, 3H), 3.76 (dd, J = 9.4, 2.4 Hz,

1H), 3.25 (ddd, J = 13.6, 5.5, 2.4 Hz, 1H), 2.87 (ddd, J = 13.6, 9.4,

5.5 Hz, 1H) 1.38 (s, 9H), 1.38 (s, 3H). ¹³C NMR (101 MHz, DMSO‑d₆) δ:

(rotamer mixture) 173.7, 155.9, 155.0, 143.8, 143.8, 140.7, 127.7,

127.1, 125.3, 120.1, 77.8, 73.1, 65.6, 61.7, 46.7, 42.6, 28.3, 18.4.

HRMS (ESI) m/z: Calculated for C₂₅H₃₁N₂O₇(M + H)⁺471.2126,

found 471.2121. Anal. Calcd for C₂₅H₃₀N₂O₇× 0.15 H₂O (0.6%) × 0.1

MeCN (0.9%): C 63.41, H 6.46, N 6.16. Found: C 63.42, H 6.41, N 6.02.

Traces of MeCN could not be removed completely even after prolonged

drying in vacuo at room temperature.

4.3.24. (2S,3S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-

((tert-butoxycarbonyl)(methyl)amino)-3-hydroxybutanoic acid (8d)

Compound 8d was prepared from 7d (0.76 g, 3.1 mmol), solid

NaHCO₃(0.97 g, 11.5 mmol), and FmocOSu (1.55 g, 4.6 mmol) as

described for 8a. After evaporation of the solvent, the residue was

puriﬁed by column chromatography on silica gel (gradient elution from

20:1 DCM/EtOH to 4:1 DCM/EtOH) to give (2S,3S)-8d (1.06 g, 73%) as

a white foam. [ ]²_D²−3.5 (c 1.0, MeOH). ¹H NMR (400 MHz, DMSO‑d₆)

δ: (rotamer mixture) 7.89 (d, J = 7.4 Hz, 2H), 7.73 (d, J = 7.4 Hz, 2H),

7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.31–7.20 (m, 1H),

4.37–4.17 (m, 3H), 4.15–3.88 (m, 2H), 3.49–3.35 (m, 2H, overlapped

with water and EtOH), 3.10 (dd, J = 14.2, 8.0 Hz, 1H), 2.80 (s, 3H),

4.3.27. (2S,3R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-

((tert-butoxycarbonyl)amino)-3-hydroxybutanoic acid (8 g)

Compound 8 g was prepared from crude compound 7 g (0.645 g,

2.8 mmol), solid NaHCO₃(0.578 g, 6.9 mmol), and FmocOSu (1.02 g,

3.0 mmol) as described for 8a. After evaporation of the solvent, the

12

E. Loza, et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

residue was puriﬁed by column chromatography on silica gel (gradient

elution from 10% EtOH in DCM to 100% EtOH). The residual ethanol

was removed by co-evaporation with acetonitrile to give (2S,3S)-8g as a

white foam (0.734 g, 58%). [ ]²_D²−0.5 (c 1.0, MeOH). ¹H NMR

(400 MHz, DMSO‑d₆) δ: (rotamer mixture, ca 9:1) 7.89 (d, J = 7.5 Hz,

2H), 7.76 (d, J = 7.5 Hz, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.42 (td,

J = 7.5, 1.0 Hz, 2H), 7.34 (td, J = 7.5, 1.0 Hz, 1H), 7.33 (td, J = 7.5,

1.0 Hz, 2H), 6.98 (d, J = 8.8 Hz, 1H), 6.69 (t, J = 5.8 Hz, 1H),

4.35–4.17 (m, 3H), 4.05 (dd, J = 8.8, 2.4 Hz, 1H), 4.00 (td, J = 6.7,

2.4 Hz, 1H), 3.13–2.76 (m, 2H), 1.37 (s, 9H). ¹³C NMR (101 MHz,

DMSO‑d₆) δ: 172.5, 156.3, 155.6, 143.9, 143.8, 140.7, 140.7, 127.6,

127.1, 125.4, 125.3, 120.1, 120.1, 77.7, 69.7, 65.6, 56.6, 46.6, 43.4,

J = 6.6 Hz, 3H). ¹³C NMR (101 MHz, D₂O) δ: 177.8, 131.0, 129.4,

129.4, 129.2, 51.5, 47.9, 38.8, 15.8. HRMS (ESI) m/z: Calculated for

C

₁₁H₁₆NO₂[M + H]⁺194.1176, found 194.1183.

4.3.30. (R)-3-(Benzylamino)butan-1-ol (11b)

To a solution of (R)-3-aminobutan-1-ol (10b) (1.76 g, 19.7 mmol) in

MeOH (30 mL) benzaldehyde (2.10 g, 19.7 mmol) was added and the

obtained mixture was stirred at room temperature for 1 h. The reaction

mixture was cooled to 0 °C, solid NaBH₄(0.75 g, 19.7 mmol) in 4

portions was added, the mixture was stirred at this temperature for

20 min, and at room temperature for 2 h. To the reaction mixture was

added water (3 mL) and the solvent was evaporated. The residue was

supplemented with water (40 mL) and extracted with DCM

(2 × 40 mL). The extract was washed with water, brine, and dried

(Na₂SO₄). After evaporation of the solvent, the residue was dried in

vacuo to give the desired product 11b as a white solid (3.25 g, 90%).

[ ]²_D²−60.5 (c 0.31, CHCl₃). ¹H NMR (400 MHz, CDCl3) δ: 7.35–7.20

(m, 5H), 3.88–3.81 (m, 1H), 3.85 (d, J = 12.7 Hz, 1H), 3.79–3.72 (m,

1H), 3.77 (d, J = 12.7 Hz, 1H), ~3.2 (br s, 2H), 2.97 (dqd, J = 8.4, 6.4,

3.4 Hz, 1H), 1.72 (ddt, J = 14.5, 6.0, 3.4 Hz, 1H), 1.53 (dtd, J = 14.5,

8.3, 3.6 Hz, 1H), 1.18 (d, J = 6.4 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ

139.8, 128.6, 128.3, 127.3, 62.5, 53.9, 51.3, 37.1, 20.4. HRMS (ESI) m/

28.2. HRMS (ESI) m/z: Calculated for

C

₂₄H₂₉N₂O₇[M

+

H]⁺

457.1969. Found: 457.1966. Anal. Calcd for C₂₄H₂₈N₂O₇× 0.15 H₂O

(0.6%) × 0.15 MeCN (1.3%): C 62.72, H 6.23, N 6.47. Found: C 62.38,

H 6.05, N 6.37. Traces of MeCN could not be removed completely even

after prolonged drying in vacuo at room temperature.

4.3.28. (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-2-((4S,5R)-

3-(tert-butoxycarbonyl)-2,2,4-trimethyloxazolidin-5-yl)acetic acid (9b)

To a solution of compound 8b (1.60 g, 3.4 mmol) in acetone/2,2-

dimethoxypropane (10 mL/10 mL) mixture at ice bath temperature a

catalytic amount of BF₃× Et₂O (0.05 mL, 0.4 mmol) was added. The

reaction mixture was stirred in the ice bath for 1 h and at room tem-

perature for 3 h, then poured into 5% aqueous solution of NaHCO₃

(30 mL) and washed with cyclohexane (10 mL) and diisopropyl ether

(2 × 10 mL). To the aqueous layer EtOAc (20 mL) was added and the

mixture was acidiﬁed with 6 N HCl to pH 3. The organic layer was

separated, washed successively with 1 M solution of KHSO₄(10 mL),

water, brine, and dried (Na₂SO₄). The solvents were evaporated, the

residue was triturated with pentane (3 × 5 mL), and dried in vacuo to

give the desired 9b as a white solid (1.40 g, 80%). [ ]²_D¹+ 3.7 (c 1.0,

MeOH). ¹H NMR (400 MHz, DMSO‑d₆) δ: 12.72 (br s, 1H), 8.01–7.91

(m, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.68 (d, J = 7.5 Hz, 2H), 7.42 (t,

J = 7.5 Hz, 2H), 7.36–7.28 (m, 2H), 4.42–4.32 (m, 1H), 4.30 (dd,

J = 10.3, 6.8 Hz, 1H), 4.23 (t, J = 7.0 Hz, 1H), 4.18 (dd, J = 10.0,

4.5 Hz, 1H), 4.04–3.88 (m, 1H), 3.96 (dd, J = 10.3, 8.5 Hz, 1H), 1.50

(s, 3H), 1.43 (s, 9H), 1.42–1.37 (m, 3H), 0.98 (d, J = 6.0 Hz, 3H). ¹³C

NMR (101 MHz, DMSO‑d₆) δ: 172.0, 155.6, 150.8, 150.7, 143.8, 143.6,

140.8, 140.8, 127.7, 127.1, 125.2, 125.2, 120.2, 120.2, 93.1, 92.8,

79.5, 79.1, 74.7, 74.3, 65.8, 53.8, 53.5, 46.7, 28.2, 28.1, 27.1, 24.9,

23.7, 14.0, 13.3. LCMS (ESI) m/z: 511.419 [M + H]⁺. HRMS (ESI) m/z:

Calculated for C₂₈H₃₄N₂NaO₇[M + Na] ⁺533.2258, found 533.2260.

Anal. Calcd for C₂₈H₃₄N₂O₇× 0.18 H₂O (0.6%): C 65.45, H 6.74, N

5.45. Found: C 65.45, H 6.77, N 5.32.

+

z: Calculated for C₁₁H₁₈NO [M + H] 180.1383, found 180.1396.

4.3.31. tert-Butyl (S)-benzyl(4-hydroxybutan-2-yl)carbamate (12a)

(S)-3-(Benzyl(tert-butoxycarbonyl)amino)butanoic acid. The water

solution of compound 11a, obtained in the preceding step, was sup-

plemented with dioxane (100 mL), cooled to 0 °C and Boc₂O (11.04 g,

50.6 mmol) followed by Et₃N (18.4 mL, 132.0 mmol) were added. The

obtained solution was stirred at 0 °C for 18 h, then the mixture was

partially evaporated (to ca of the half of the initial volume), acidiﬁed

with 2 M HCl to pH = 4, and extracted with EtOAc (2 × 100 mL). The

extracts were combined, dried (Na₂SO₄), and evaporated. The residue

was puriﬁed by column chromatography on silica gel (eluent DCM/

MeOH, 40:1) to give (S)-3-(benzyl(tert-butoxycarbonyl)amino)butanoic

acid as a white solid (9.70 g, 68% in two steps from 10a). [ ]²_D²+31.1°

(c 1.0, MeOH). ¹H NMR (400 MHz, CDCl₃) δ: 9.56 (br s, 1H), 7.34–7.19

(m, 5H), 4.72–3.96 (m, 3H), 2.73 (dd. J = 15.5, 6.7 Hz, 1H), 2.47 (dd.

J = 15.5, 7.2 Hz, 1H), 1.45 (s, 9H), 1.18 (d, J = 6.6 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ: 177.2, 155.8, 139.3, 128.5, 127.6, 127.2, 80.4,

50.2, 49.3, 40.0, 28.5, 19.1. HRMS (ESI) m/z: Calculated for

C₁₆H₂₃NNaO₄[M + Na]⁺316.1519, found 316.1522.

tert-Butyl (S)-benzyl(4-hydroxybutan-2-yl)carbamate (12a).

To a solution of above prepared (S)-3-(benzyl(tert-butoxycarbonyl)

amino)butanoic acid (8.50 g, 29.0 mmol) in THF (150 mL) at −72 °C

under argon atmosphere BH₃-THF complex (1 M solution in THF,

87.0 mL, 87.0 mmol) was added dropwise. The reaction mixture was

allowed to warm up to room temperature and stirred for 20 h. To the

mixture MeOH (50 mL) was added, stirred for 30 min, and the solvent

was evaporated. The residue was partitioned between water (100 mL)

and EtOAc (100 mL), and the aqueous layer was extracted with EtOAc

(2 × 100 mL). The organic extracts were combined, washed succes-

sively with water (10 mL), brine (50 mL), and dried (Na₂SO₄). After

evaporation of the solvent, the residue was puriﬁed by column chro-

matography on silica gel (gradient elution from 5:1 petroleum ether/

EtOAc to 1:1 petroleum ether/EtOAc) to give 12a as a white solid

(7.12 g, 88%). [ ]²_D²+ 17.6 (c 1.0, MeOH). ¹H NMR (400 MHz, CDCl₃)

δ: 7.37–7.17 (m, 5H), 4.53 (br s, 1H), 4.41 (d, J = 16.1 Hz, 1H), 4.18

(d, J = 16.1 Hz, 1H), 3.59–3.34 (m, 3H), 1.72–1.59 (m, 1H), 1.59–1.35

(m, 1H), 1.39 (s, 9H), 1.13 (d, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz,

CDCl₃) δ: 157.5, 139.9, 128.4, 126.9, 126.8, 80.6, 58.9, 47.4, 45.9,

4.3.29. (S)-3-(Benzylamino)butanoic acid (11a)

To a solution of (S)-3-aminobutyric acid 10a (5.00 g, 48.5 mmol) in

10:7 (v:v) mixture of MeOH and H₂O (170 mL), benzaldehyde (5.15 g,

48.5 mmol) followed by a solution of NaOH

(1.94 g, 48.5 mmol) in water (40 mL) were added and the obtained

solution was stirred at room temperature for 1 h. The reaction mixture

was cooled to 0 °C, solid NaBH₄(0.74 g, 19.4 mmol) in 4 portions was

added, and the mixture was allowed to warm up to room temperature.

The mixture was partially evaporated to ca of the half of the initial

volume, acidiﬁed with 2 M HCl to pH = 7, and washed successively

with EtOAc (3 × 70 mL) and DCM (2 × 100 mL). The aqueous phase

was utilized in the next step of the synthesis without the isolation of the

product. To obtain analytically pure sample of the desired product 11a,

an aliquot from the aqueous phase was puriﬁed by reverse phase

column chromatography (gradient elution from 0% MeCN in water to

70% MeCN in water). After evaporation of the solvent, the residue was

dried in vacuo to give 11a as a white solid. [ ]²_D²+ 33.5 (c 1.0, MeOH).

¹H NMR (400 MHz, D₂O) δ: 7.53–7.45 (m, 5H), 4.28 (d, J = 13.3 Hz,

1H), 4.21 (d, J = 13.3 Hz, 1H), 3.56 (sextet, J = 6.6 Hz, 1H), 2.56 (dd,

J = 16.7, 6.3 Hz, 1H), 2.53 (dd, J = 16.7, 6.7 Hz, 1H), 1.38 (d,

37.7, 28.4, 19.7. HRMS (ESI) m/z: Calculated for

C₁₆H₂₅NNaO₃

+

[M + Na] 302.1727, found 302.1730.

4.3.32. tert-Butyl (R)-benzyl(4-hydroxybutan-2-yl)carbamate (12b)

To a solution of compound 11b (3.00 g, 16.7 mmol) in DCM (50 mL)

13

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Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

were added Et₃N (2.6 mL, 18.7 mmol) followed by a solution of Boc₂O

(3.84 g, 17.6 mmol) in DCM (15 mL) and the resulting mixture was

stirred at room temperature for 18 h. The mixture was washed suc-

cessively with cold 0.5 M aqueous HCl solution, brine, and dried

(Na₂SO₄). After evaporation of the solvent, the residue was dried in

vacuo to give the desired product 12b as a white solid (3.90 g, 80%).

[ ]²_D²−18.5 (c 1.0, MeOH). ¹H NMR (400 MHz, CDCl₃) δ: 7.37–7.17 (m,

5H), 4.54 (br s, 1H), 4.41 (d, J = 16.1 Hz, 1H), 4.17 (d, J = 16.1 Hz,

1H), 3.59–3.34 (m, 3H), 1.71–1.60 (m, 1H), 1.57–1.44 (m, 1H), 1.39 (s,

9H), 1.13 (d, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ: 157.5,

139.9, 128.4, 126.9, 126.8, 80.6, 58.9, 47.4, 45.9, 37.7, 28.4, 19.7.

290.0400, found 290.0414.

4.3.36. tert-Butyl benzyl(3-iodopropyl)carbamate (13c)

Compound 13c was prepared from 12b (4.04 g, 15.2 mmol), tri-

phenylphosphine (5.31 g, 20.2 mmol), iodine (5.14 g, 20.2 mmol), and

imidazole (1.81 g, 26.6 mmol) as described for 13a. Puriﬁcation by

column chromatography on silica gel (eluent petroleum ether/EtOAc to

9:1) aﬀorded 13c as a slightly yellowish oil (4.79 g, 83%). ¹H NMR

(400 MHz, DMSO‑d₆) δ: (rotamer mixture) 7.38–7.31 (m, 2H),

7.30–7.19 (m, 3H), 4.45 and 4.38 (s and s, altogether 2H), 3.23–3.13

(m, 4H), 1.98–1.87 (m, 2H), 1.43 and 1.38 (br s and br s, 5H and 4H).

¹³C NMR (101 MHz, DMSO‑d₆) δ: (rotamer mixture) 155.0, 153.1,

138.6, 137.4, 128.5, 128.5, 127.4, 127.2, 127.1, 79.0, 66.4, 51.7, 49.5,

47.1, 44.5, 31.7, 28.1, 23.9, 21.8, 4.8. HRMS (ESI) m/z: Calculated for

+

HRMS (ESI) m/z: Calculated for C₁₆H₂₅NNaO₃[M + Na] 302.1727,

found 302.1735.

C

₁₀H₁₅NI [M−COO^tBu + 2H]⁺276,0244, found 276.0246.

4.3.33. tert-Butyl benzyl(3-hydroxypropyl)carbamate (12c)

The title compound was obtained according to the literature pro-

cedure.⁴⁴(Boc)₂O (13.21 g, 60.5 mmol) was added to a stirred solution

of commercial 11c (10.00 g, 60.5 mmol) in 5:1 (v:v) mixture of DCM

and 1.0 M aqueous NaOH solution at 0 °C. The resultant mixture was

allowed to warm to room temperature and stirred for 16 h. The reaction

mixture was washed with water (200 mL) and dried (Na₂SO₄). After

evaporation of the solvent, the residue was puriﬁed by column chro-

matography on silica gel (gradient elution from 9:1 petroleum ether/

EtOAc to 2:1 petroleum ether/EtOAc) to give 12c as a colorless oil

(10.54 g, 66%). ¹H NMR (400 MHz, CDCl₃) δ: 7.35–7.30 (m, 2H),

7.29–7.20 (m, 3H), 4.39 (s, 2H), 3.73 (t, J = 6.6 Hz, 1H), 3.56 (q,

J = 6.1 Hz, 2H), 3.44–3.23 (m, 2H), 1.69–1.57 (m, 2H), 1.47 (s, 9H).

¹³C NMR (101 MHz, CDCl₃) δ: 157.2, 138.3, 128.7, 127.4, 80.7, 58.5,

50.6, 42.3, 30.3, 28.5. LCMS (ESI) m/z: 266.01 [M + H]⁺. The spectra

were in agreement with those reported in the literature.⁴⁴

4.3.37. Benzyl (3R,5R,6S)-3-((S)-3-(benzyl(tert-butoxycarbonyl)amino)

butyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (14a)

To a solution of compound (R,S)-4 (3.75 g, 9.7 mmol) and HMPA

(30 mL) in THF (350 mL) at –75 °C under argon atmosphere NaHMDS

(2 M solution in THF, 5.4 mL, 10.8 mmol) was added. After stirring at

this temperature for 10 min, a solution of compound 13a (5.01 g,

12.9 mmol) in THF (15 mL) was added and the stirring was continued

at −55 °C for 2 h. The reaction mixture was gradually (during 3 h)

warmed to room temperature, whereupon almost all of (R,S)-4 was

consumed (TLC and LCMS control). The reaction mixture was cooled to

0 °C, supplemented with brine (150 mL) and extracted with ethyl

acetate (3 × 150 mL). The organic extracts were combined, dried

(Na₂SO₄), and solvents were evaporated. The residue was puriﬁed by

column chromatography on silica gel (gradient elution from 10:1 pet-

roleum ether/EtOAc to 5:1 petroleum ether/EtOAc) to give 14a as a

white solid (4.52 g, 72%). [ ]²_D²+ 4.9 (c 1.0, CH₂Cl₂). ¹H NMR

(400 MHz, DMSO‑d₆) δ: (3:2 mixture of rotamers) 7.43–6.99 (m,

16.8H), 6.72 (d, J = 7.5 Hz, 1.2H), 6.56 (d, J = 7.5 Hz, 0.8H), 6.52 (d,

J = 7.6 Hz, 1.2H), 6.30–6.12 (m, 1H), 5.31 (d, J = 2.6 Hz, 0.6H), 5.26

(d, J = 2.6 Hz, 0.4H), 5.09 (d, J = 12.7 Hz, 0.4H), 5.07 (d,

J = 12.7 Hz, 0.4H), 4.98 (d, J = 13.0 Hz, 0.6H), 4.90 (d, J = 13.0 Hz,

0.6H), 4.77 (dd, J = 8.4, 6.1 Hz, 1H), 4.48–3.44 (m, 3H), 2.16–1.53 (m,

4H), 1.53–1.18 (m, 9H), 1.06 (d, J = 6.0 Hz, 1.8H), 0.96 (d, J ~ 6 Hz,

1.2H). ¹³C NMR (101 MHz, DMSO‑d₆) δ (3:2 mixture of rotamers)

168.3, 168.1, 154.9, 153.6, 153.0, 139.9, 136.4, 136.2, 136.0, 135.9,

134.7, 134.6, 128.4, 128.2, 128.1, 128.1, 128.0, 127.9, 127.8, 127.6,

127.5, 127.4, 127.4, 127.2, 127.1, 126.6, 126.3, 79.0, 78.7, 77.9, 77.8,

67.0, 66.5, 59.9, 56.9, 56.6, 52.4, 50.9, 46.3, 31.2, 30.5, 28.0, 19.1,

4.3.34. tert-Butyl (S)-benzyl(4-iodobutan-2-yl)carbamate (13a)

To a solution of triphenylphosphine ((8.89 g, 33.9 mmol) in DCM

(500 mL) at 0 °C was added iodine (8.61 g, 33.9 mmol), the mixture was

stirred for 15 min and imidazole (3.04 g, 44.6 mmol) was added. The

obtained mixture was stirred at 0 °C for 15 min and then 12a (7.12 g,

25.5 mmol) was added. The ice bath was removed, the reaction mixture

was stirred at room temperature for 1 h, and supplemented with water

(50 mL). The organic layer was separated and the aqueous phase was

extracted with DCM (2 × 50 mL). The organic phases were combined,

washed with a saturated aqueous Na₂S₂O₃solution (50 mL), brine

(100 mL), and dried (Na₂SO₄). After evaporation of the solvent, the

residue was puriﬁed by column chromatography on silica gel (gradient

elution from 20:1 petroleum ether/EtOAc to 10:1 petroleum ether/

EtOAc) to give 13a as a white solid (4.49 g, 45%). [ ]²_D²17.9 (c 1.0,

MeOH). ¹H NMR (400 MHz, CDCl₃) δ: (2 rotamer mixture, ca 1:1)

7.36–7.21 (m, 5H), 4.61–4.22 (m, 2H), 4.20–4.00 and 3.89 (m and br s,

0.5H and 0.5H), 3.23–2.81 (m, 2H), 2.28–2.07 (m, 1H), 2.02–1.76 (m,

1H), 1.51 and 1.40 (br s and br s, 4.5H and 4.5H), 1.10 (d, J = 6.5 Hz,

3H). HRMS (ESI) m/z: Calculated for C₁₁H₁₇NI [M−COO^tBu + 2H]⁺

290.0400, found 290.0413.

+

18.8. HRMS (ESI) m/z: Calculated for C₄₀H₄₅N₂O₆[M + H]

649.3272, found 649.3275.

4.3.38. Benzyl (3R,5R,6S)-3-((R)-3-(benzyl(tert-butoxycarbonyl)amino)

butyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (14b)

Compound 14b (0.98 g, 58%) was prepared as a white solid from

(R,S)-4 (1.00 g, 2.6 mmol), HMPA (9.9 mL), NaHMDS (2 M solution in

THF, 1.4 mL, 2.8 mmol), and compound 13b (1.34 g, 3.4 mmol) as

described for 14a. [ ]²_D²−10.3 (c 1.0, CH₂Cl₂). ¹H NMR (400 MHz,

DMSO‑d₆) δ: (3:2 mixture of rotamers) 7.40–7.03 (m, 16.8H), 6.70 (d,

J = 7.5 Hz, 1.2H), 6.54 (d, J = 7.5 Hz, 0.8H), 6.50 (d, J = 7.6 Hz,

1.2H), 6.07 (br s, 1H), 5.29 (d, J = 2.6 Hz, 0.6H), 5.23 (d, J = 2.6 Hz,

0.4H), 5.16 (d, J = 12.4 Hz, 0.4H), 4.98 (d, J = 12.4 Hz, 0.4H), 4.98

(d, J = 13.0 Hz, 0.6H), 4.88 (d, J = 13.0 Hz, 0.6H), 4.79–4.68 (m, 1H),

4.53–3.93 (m, 2.6H), 3.93–3.56 (m, 0.4H), 2.13–1.65 (m, 3H),

1.61–1.16 (m, 1H), 1.39 (br s, 5.4H), 1.29 (br s, 3.6H), 1.08 (d,

J = 6.5 Hz, 1.8H), 0.98 (d, J = 6.5 Hz, 1.2H). ¹³C NMR (101 MHz,

DMSO‑d₆) δ (3:2 mixture of rotamers) 168.2, 168.1, 155.3, 154.9,

153.6, 153.0, 140.0, 139.7, 136.4, 136.2, 136.1, 135.9, 134.6, 134.6,

128.5, 128.3, 128.2, 128.0, 128.0, 127.9, 127.8, 127.6, 127.5, 127.5,

127.5, 127.3, 127.1, 126.8, 126.6, 126.3, 78.9, 78.0, 77.8, 67.0, 66.5,

60.0, 59.9, 57.0, 56.8, 52.6, 51.4, 48.0, 46.4, 31.8, 30.9, 30.7, 28.0,

4.3.35. tert-Butyl (R)-benzyl(4-iodobutan-2-yl)carbamate (13b)

Compound 13b was prepared from 12b (3.90 g, 13.5 mmol), tri-

phenylphosphine (4.73 g, 18.0 mmol), iodine (4.57 g, 18.0 mmol), and

imidazole (1.62 g, 23.8 mmol) as described for 13a. Puriﬁcation by

column chromatography on silica gel (gradient elution from 20:1 pet-

roleum ether/EtOAc to 10:1 petroleum ether/EtOAc) aﬀorded 13b as a

white solid (2.01 g, 38%). [ ]²_D²−20.3 (c 1.0, MeOH). ¹H NMR

(400 MHz, CDCl₃) δ: (2 rotamer mixture, ca 1:1) 7.36–7.20 (m, 5H),

4.61–4.23 (m, 2H), 4.19–4.00 and 3.89 (m and br s, 0.5H and 0.5H),

3.23–2.79 (m, 2H), 2.28–2,06 (m, 1H), 2.02–1.76 (m, 1H), 1.51 and

1.41 (br s and br s, 4.5H and 4.5H), 1.10 (d, J = 6.3 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ: (2 rotamer mixture, ca 1:1) 155.5, 139.7, 128.8,

128.5, 127.9, 127.6, 127.1, 80.2, 53.5, 48.4, 40.6, 39.2, 28.6, 18.9, 2.9.

HRMS (ESI) m/z: Calculated for C₁₁H₁₇NI [M−COO^tBu + 2H]⁺

14

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+

19.2, 18.7. HRMS (ESI) m/z: Calculated for C₄₀H₄₅N₂O₆[M + H]

J = 6.6 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆) δ: 173.9, 156.1, 155.0,

649.3272, found 649.3262.

143.8, 143.8, 140.7, 127.6, 127.1, 125.3, 125.3, 120.1, 77.3, 65.6,

53.6, 46.7, 45.3, 32.7, 28.3, 27.6, 21.1. HRMS (ESI) m/z: Calculated for

+

C

₂₆H₃₃N₂O₆[M + H]

469.2333, found 469.2338. Anal. Calcd for

4.3.39. (3R,5R,6S)-3-(3-(Benzyl(tert-butoxycarbonyl)amino)propyl)-3-

C

₂₆H₃₂N₂O₆× 0.05 MeCN (0.4%) × 0.1 H₂O (0.4%): C 66.36, H 6.90,

methyl-2-oxo-5,6-diphenylmorpholine-4-carboxylate (14c)

N 6.08. Found: C 66.28, H 6.83, N 6.11. The presence of MeCN (ca.

0.05 eq, 0.4%) was detected by ¹H NMR. Traces of MeCN could not be

removed after prolonged drying in vacuo at room temperature.

KHMDS (1 M solution in THF, 10.7 mL, 10.8 mmol) slowly (over

20 min) was added to a solution of compound (2R)(R,S)-5 (3.30 g,

8.2 mmol), 13c (6.79 g, 18.1 mmol), and HMPA (3 mL) in THF (30 mL)

at –78 °C under argon atmosphere. After stirring at this temperature for

4 h, saturated aqueous NH₄Cl solution (10 mL) was added and the

mixture was allowed to warm up to room temperature. The mixture was

supplemented with EtOAc (50 mL) and the layers were separated. The

organic layer was washed with saturated aqueous Na₂S₂O₃solution

(2 × 10 mL), brine (10 mL), dried (Na₂SO₄), and the solvents were

evaporated. The residue was puriﬁed by column chromatography on

silica gel (gradient eluent from 10:1 petroleum ether/EtOAc to 3:2

petroleum ether/EtOAc) to give 14c as a colorless foam (4.40 g, 82%).

[ ]²_D⁰−28.1 (c 1.0, CHCl₃). ¹H NMR (400 MHz, DMSO‑d₆) δ: (rotamer

mixture) 7.39–7.03 (m, 18H), 6.98–6.66 (m, 2H), 6.29–6.10 (m, 1H),

5.72–5.40 (m, 1H), 5.23–4.95 (m, 2H), 4.46–4.24 (m, 2H), 3.27–2.94

4.3.41. (2R,5R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-

((tert-butoxycarbonyl)amino)hexanoic acid (16b)

Compound 15b was prepared from 14b (5.41 g, 8.3 mmol), lithium

chunks (1.72 g, 246.7 mmol), and NH₄Cl (11.60 g, 217 mmol) as de-

scribed for 15a. Crude (2R,5R)-2-amino-5-((tert-butoxycarbonyl)

amino)hexanoic acid (15b) (12.1 g) was obtained as a mixture with

inorganic salts. The recorded ¹H NMR was not informative, however,

the presence of the desired product 15b (in a form of lithium salt) was

supported by LCMS (ESI) m/z: 247.3 [M + H]⁺. The obtained solid was

used in the next step without further puriﬁcation.

Compound 16b was prepared as a white solid (1.05 g, 27% in two

steps from 14b) from crude 15b (12.1 g), FmocOSu (3.59 g,

10.7 mmol), and solid NaHCO₃(1.07 g, 12.7 mmol) as described for

16a. [ ]²_D²+ 7.1 (c 1.0, MeOH). ¹H NMR (400 MHz, DMSO‑d₆) δ: 12.54

(br s, 1H) 7.89 (d, J = 7.4 Hz, 2H), 7.72 (d, J = 7.4 Hz, 2H), 7.62 (d,

J = 8.0 Hz, 1H), 7.42 (td, J = 7.4, 1.0 Hz, 2H), 7.33 (td, J = 7.4,

1.1 Hz, 2H), 6.68 (d, J = 8.3 Hz, 1H), 4.32–4.17 (m, 3H), 3.95–3.81 (m,

1H), 3.46–3.36 (m, overlapped with water, 1H), 1.79–1.65 (m, 1H),

1.61–1.48 (m, 1H), 1.47–1.30 (m, 2H) 1.37 (s, 9H), 1.00 (d, J = 6.6 Hz,

3H). ¹³C NMR (101 MHz, DMSO‑d₆) δ: 173.9, 156.1, 155.1, 143.8,

143.8, 140.7, 140.7, 127.6, 127.1, 127.1, 125.3, 125.3, 120.1, 77.3,

(m, 2H), 2.47–1.99 (m, 2H), 1.86–1.47 (m, 5H), 1.47–1.21 (m, 9H). ¹³

C

NMR (101 MHz, DMSO‑d₆) δ (rotamer mixture) 171.2, 171.0, 155.1,

154.7, 152.9, 138.7, 136.2, 136.0, 135.3, 128.4, 128.4, 128.1, 128.1,

127.8, 127.3, 127.2, 127.0, 125.9, 78.9, 78.3, 66.6, 63.1, 60.1, 50.0,

49.7, 46.3, 34.9, 28.0, 23.9, 23.5, 23.0, 22.8. HRMS (ESI) m/z:

Calculated for C₄₀H₄₄N₂NaO₆[M + Na] ⁺671.3092, found 671.3087.

4.3.40. (2R,5S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-

((tert-butoxycarbonyl)amino)hexanoic acid (16a)

Ammonia (about 150 mL) was condensed in a 250 mL ﬂask at

−75 °C, then lithium chunks (1.30 g, 187 mmol) were added. The re-

sulting dark blue solution was stirred for 30 min and then a solution of

compound 14a (4.50 g, 6.9 mmol) in THF (50 mL) was added dropwise.

The reaction mixture was stirred at −75 °C for 1 h and then carefully

quenched with solid NH₄Cl (9.65 g, 180 mmol) at this temperature. The

mixture was supplemented with water (5 mL), stirred until ammonia

evaporated, and then another portion of water (30 mL) was added. The

mixture was washed with Et₂O (2 × 30 mL), the water layer was

concentrated at room temperature, and the residue was dried in vacuo

to give (2R,5S)-2-amino-5-((tert-butoxycarbonyl)amino)hexanoic acid

(15a) as a solid material (10.9 g) in a mixture with inorganic salts. The

recorded ¹H NMR was not informative, however, the presence of the

desired product 15a (in a form of lithium salt) was supported by LCMS

(ESI) m/z: 247.6 [M + H]⁺. The obtained solid was used in the next

step without further puriﬁcation.

65.6, 54.9, 54.1, 46.7, 45.7, 33.1, 28.3, 27.9, 21.0. HRMS (ESI) m/z:

+

Calculated for C₂₆H₃₃N₂O₆[M + H]

469.2333, found 469.2337.

Anal. Calcd for C₂₆H₃₂N₂O₆× 0.13 DCM (2.1%): C 65.46, H 6.75, N

5.84. Found: C 65.39, H 6.75, N 5.79. The presence of DCM (ca. 0.13 eq,

2.1%) was detected by ¹H NMR. Traces of solvent could not be removed

even after prolonged drying in vacuo at room temperature.

4.3.42. (R)-2-Amino-5-((tert-butoxycarbonyl)amino)-2-methylpentanoic

acid (15c)

Compound 15c was prepared from 14c (4.00 g, 6.2 mmol), lithium

chunks (1.33 g, 191.1 mmol), and NH₄Cl (9.89 g, 185 mmol) as de-

scribed for 15a.The obtained crude 15c was puriﬁed by reverse phase

column chromatography (gradient elution from 0% MeCN in water to

50% MeCN in water) to give 15c as an oﬀ-white solid (0.46 g, 29%).

[ ]²_D⁰−3.4 (c 0.92, MeOH/H₂O, 1:5). ¹H NMR (400 MHz, DMSO‑d₆) δ:

7.48 (br s, 3H), 6.79 (t, J = 5.7 Hz, 1H), 2.84 (q, J = 6.5 Hz, 2H),

1.65–1.54 (m, 1H), 1.54–1.40 (m, 2H), 1.40–1.24 (m, 1H), 1.36 (s, 9H),

1.22 (s, 3H). ¹³C NMR (101 MHz, DMSO‑d₆) δ: 172.1, 155.6, 77.3, 59.7,

Crude solid 15a (10.9 g) from above was mixed with water

(100 mL). The formed suspension was cooled in an ice bath and solid

NaHCO₃(1.46 g, 17.3 mmol) was added to this suspension, followed by

a solution of FmocOSu (7.25 g, 21.5 mmol) in dioxane (40 mL). The

reaction mixture was stirred at room temperature for 36 h, supple-

mented with water (200 mL), and washed with Et₂O (200 mL). The

aqueous layer was cooled in an ice bath and treated with 1 M aqueous

HCl solution until the pH 5. The mixture was extracted with EtOAc

(3 × 200 mL), the organic extracts were combined, washed succes-

sively with water, brine, and dried (Na₂SO ₄). After evaporation of the

solvent, the residue (1.6 g) was puriﬁed by column chromatography on

silica gel (gradient elution from 25:1 DCM/MeOH to 15:1 DCM/

MeOH), followed by reverse phase column chromatography (gradient

elution from 5% MeCN in water to 40% MeCN in water) to give 16a as a

white solid (0.71 g, 22% in two steps from 14a). [ ]²_D²−7.4 (c 1.0,

MeOH). ¹H NMR (400 MHz, DMSO‑d₆) δ: 12.54 (br s, 1H) 7.89 (d,

J = 7.4 Hz, 2H), 7.72 (d, J = 7.4 Hz, 2H), 7.57 (d, J = 8.0 Hz, 1H),

7.42 (td, J = 7.4, 1.0 Hz, 2H), 7.33 (td, J = 7.4, 1.1 Hz, 2H), 6.60 (d,

J = 8.2 Hz, 1H), 4.33–4.16 (m, 3H), 3.99–3.86 (m, 1H), 3.50–3.36 (m,

overlapped with water, 1H), 1.78–1.64 (m, 1H), 1.63–1.50 (m, 1H),

1.46–1.33 (m, 2H, overlapped with tBu) 1.38 (s, 9H), 0.99 (d,

40.1, 35.2, 28.3, 24.5, 23.2. HRMS (ESI) m/z: Calculated for

+

C

₁₁H₂₃N₂O₄[M + H] 247.1652, found 247.1653.

4.3.43. (R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-((tert-

butoxycarbonyl)amino)-2-methylpentanoic acid (16c)

A solution of 15c (443 mg, 1.80 mmol) in 5% aqueous NaHCO₃

solution (24 mL) was added to a solution of FmocOSu (770 mg,

2.28 mmol) in THF (12 mL) and stirred at room temperature for 16 h.

The reaction mixture was supplemented with another amount of

FmocOSu (237 mg, 0.70 mmol), and the stirring was continued for 6 h.

The mixture was evaporated to ca 2/3 of the initial volume, washed

with Et₂O (2 × 10 mL), and acidiﬁed by 2 M aqueous HCl solution until

pH 3. The mixture was extracted with EtOAc (3 × 30 mL), the organic

extracts were combined, washed successively with 1 M aqueous KHSO₄

solution, water, brine, and dried (Na₂SO ₄). After evaporation of sol-

vent, the residue was puriﬁed by reverse phase column chromato-

graphy (gradient elution from 20% MeCN in water to 90% MeCN in

water) to give a white solid. The solid was suspended in pentane (6 mL),

15

E. Loza, et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

stirred for 16 h, ﬁltered, rinsed with pentane (3 × 3 mL), and dried in

vacuo to give the desired product 16c as white crystals (640 mg, 76%).

Mp: 77.3 °C. [ ]²_D⁰−6.6 (c 0.625, DCM). ¹H NMR (400 MHz, DMSO‑d₆)

δ: (2 rotamer mixture, ca 9:1) 12.35 (br s, 1H), 7.88 (d, J = 7.5 Hz, 2H),

7.71 (d, J = 7.5 Hz, 2H), 7.41 (td, J = 7.5, 1.0 Hz, 2H), 7.41 (br s, 1H),

7.34 (td, J = 7.5, 1.0 Hz, 2H), 6.77 and 6.42 (t and br s, J ~ 5.3 Hz,

0.9H and 0.1H), 4.30–4.16 (m, 3H), 2.94–2.82 (m, 2H), 1.81–1.70 (m,

1H), 1.70–1.58 (m, 1H), 1.44–1.22 (m, 2H), 1.36 (s, 9H), 1.32 (s, 3H).

¹³C NMR (101 MHz, DMSO‑d₆) δ: (2 rotamer mixture, ca 9:1) 175.3,

155.5, 154.8, 143.9, 143.8, 140.7, 127.6, 127.1, 125.3, 120.1, 77.4,

65.3, 58.1, 46.7, 34.1, 28.3, 23.9, 22.4. HRMS (ESI) m/z: Calculated for

column chromatography on silica gel (eluent CHCl₃/MeOH, 20:1) to

give (R)-21 as a glass-like material (262 mg, 44%). [ ]²_D²−8.95 (c 1.56,

CHCl₃). ¹H NMR spectrum (400 MHz, CDCl₃): δ (rotamer mixture, 9:1,

major rotamer) 7.76 (d, J = 7.5 Hz, 2H), 7.61 (d, J≈7.5 Hz, 1H), 7.59

(d, J≈7.5 Hz, 1H), 7.39 (t, J = 7.5 Hz, 2H), 7.31 (td, J = 7.5, 1.1 Hz,

2H), 5.94 (d, J = 7.1 Hz, 1H), 4.47 (q, J = 7.1 Hz, 1H), 4.44–4.34 (m,

2H), 4.22 (t, J = 6.9 Hz, 1H), 3.58–3.44 (m, 2H), 2.05–1.93 (m, 1H),

1.93–1.81 (m, 1H), 1.78–1.63 (m, 2H), 1.24 (s, 9H). ¹³C NMR

(101 MHz, CDCl₃): δ 174.6, 156.3, 144.0, 143.9, 141.4, 127.8, 127.2,

125.2, 120.1, 74.9, 67.2, 61.7, 53.6, 47.3, 30.6, 27.5, 25.6. LCMS (ESI)

m/z: [M + H]⁺412.27. Anal. Calcd for C₂₄H₂₉NO₅× 0.1 CHCl₃

(2.8%) × 0.25 H₂O (1.1%): C 67.64, H 6.97, N 3.27. Found: C 67.64, H

6.98, N 3.30. The presence of CHCl₃(ca. 0.1 eq, 2.8%) was detected by

¹H NMR. Traces of solvent could not be removed even after prolonged

drying in vacuo at room temperature.

C

₂₆H₃₂N₂NaO₆[M + Na]⁺491.2153, found 491.2162. Anal. Calcd for

₂₆H₃₂N₂O₆× 0.35 H₂O (1.3%): C 65.76, H 6.94, N 5.90. Found: C

65.77, H 6.89, N 5.87.

4.3.44. 1-(tert-Butoxy)-3-iodopropane (18)

A

solution of 1-bromo-3-(tert-butoxy)propane (17) (3.59 g,

4.3.47. (2S,3S)-2-(((Benzyloxy)carbonyl)amino)-4-((tert-butoxycarbonyl)

amino)-3-hydroxybutanoic acid (23)

18.4 mmol, 1 equiv) and NaI (3.44 g, 23.0 mmol, 1.25 eq) in dry

acetone (100 mL) was stirred at room temperature for 24 h, whereupon

the mixture was partially concentrated (up to ca 20% of the initial

volume). The residue was partitioned between brine (50 mL) and EtOAc

(50 mL), the aqueous phase was extracted with EtOAc (2 × 50 mL) and

the combined organic extracts were washed sequentially with aqueous

saturated sodium thiosulfate, brine, and dried (Na₂SO₄). Volatiles were

evaporated and the residue was dried in vacuo to give 18 as a colorless

oil (3.06 g, 69%). ¹H NMR spectrum (400 MHz, CDCl₃): δ 3.41 (t,

J = 5.8 Hz, 2H), 3.29 (t, J = 6.6 Hz, 2H), 2.04–1.97 (m, 2H), 1.19 (s,

9H). ¹³C NMR (101 MHz, CDCl₃): δ 72.9, 60.9, 34.2, 27.8, 4.6. GCMS,

Solid NaOH (0.89 g, 22.3 mmol) was added portionwise to a sus-

pension of compound 22¹¹(3.96 g, 7.4 mmol) in water (70 mL) at room

temperature. A solution of Na₂EDTA·2H₂O (4.16 g, 11.2 mmol) in water

(70 mL) was added to the dark blue solution, the mixture was stirred at

room temperature for 4 h whereupon it was cooled to 0 °C (crushed ice)

and a solution of CbzOSu (3.52 g, 14.1 mmol) in dioxane (120 mL) was

added, followed by solid Na₂CO₃(1.97 g, 18.6 mmol). The reaction

mixture was stirred at room temperature for 16 h, supplemented with

water (100 mL), and washed with Et₂O (3 × 120 mL). The mixture was

supplemented with EtOAc (120 mL) and acidiﬁed with conc. HCl to pH

3. The organic layer was separated, washed successively with 1 M

aqueous solution of KHSO₄(60 mL), water (60 mL), brine (60 mL), and

dried (Na₂SO₄). After evaporation of the solvent, the obtained solid

material was triturated with MeCN (50 mL) at room temperature for

16 h, and ﬁltered. The precipitate was dried in vacuo over P₂O₅to give

compound 23 as a white solid (4.80 g, 88%). [ ]²_D¹+ 9.6 (c 1.0, MeOH).

¹H NMR (400 MHz, DMSO‑d₆) δ: 12.60 (br s, 1H), 7.40–7.27 (m, 6H),

6.62 (t, J = 5.6 Hz, 1H), 5.07 (br s, 1H), 5.04 (d, J = 12.5 Hz, 1H), 5.03

(d, J = 12.5 Hz, 1H), 4.10 (dd, J = 9.0, 4.5 Hz, 1H), 3.87–3.80 (m,

1H), 3.09 (dt, J = 13.6, 5.6 Hz, 1H), 3.01 (dt, J = 13.6, 6.6 Hz, 1H),

1.37 (s, 9H). ¹³C NMR (101 MHz, DMSO‑d₆) δ: 171.7, 156.1, 155.7,

136.9, 128.3, 127.8, 127.7, 77.8, 69.9, 65.5, 57.5, 43.0, 28.2. HRMS

(ESI) m/z: Calculated for C₁₇H₂₄N₂NaO₇[M + Na]⁺391.1476, found

391.1479.

m/z: 242.0 M⁺

,

227.0 [M−CH₃]⁺

,

185.9 [M−C₄H₈]⁺

, 168.9

[M−C₄H₉O]⁺, 57.1 [C₄H₉]⁺

.

4.3.45. Benzyl

(3R,5R,6S)-3-(3-(tert-butoxy)propyl)-2-oxo-5,6-

diphenylmorpholine-4-carboxylate (19)

Compound 19 was prepared as described for 14a from (R,S)-4

(1.28 g, 3.3 mmol), HMPA (13 mL), 2 M solution of NaHMDS in THF

(1.82 mL, 3.6 mmol), and compound 18 (1.00 g, 4.1 mmol) as a white

solid (0.38 g, 23%). [ ]²_D⁰46.6 (c 1.63, CHCl₃). ¹H NMR spectrum

(400 MHz, DMSO‑d₆): δ (rotamer mixture, 3:2) 7.43–7.03 (m, 11.8H),

6.69 (d, J = 7.5 Hz, 1.2H), 6.55 (d, J = 7.5 Hz, 0.8H), 6.51 (d,

J = 7.5 Hz, 1.2H), 6.30–6.26 (m, 1H), 5.32 (d, J = 2.9 Hz, 0.6H), 5.25

(d, J = 2.9 Hz, 0.4H), 5.16 (d, J = 12.4 Hz, 0.4H), 5.02 (d,

J = 12.4 Hz, 0.4H), 4.99 (d, J = 13.0 Hz, 0.6H), 4.88 (d, J = 13.0 Hz,

0.6H), 4.83–4.77 (m, 1H), 3.39 (t, J = 6.8 Hz, 1.2H), 3.30 (t,

J = 6.7 Hz, 0.8H), 2.26–2.06 (m, 2H), 1.71–1.49 (m, 2H), 1.14 (s,

5.4H), 1.10 (s, 3.6H). ¹³C NMR (101 MHz, DMSO‑d₆): δ (rotamer

mixture, 3:2) 168.5, 168.3, 153.6, 153.1, 136.5, 136.3, 136.1, 136.0,

134.8, 134.7, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.9, 127.8,

127.6, 127.5, 127.5, 127.4, 127.3, 127.1, 126.6, 126.3, 78.0, 77.8,

72.1, 72.0, 67.0, 66.5, 60.1, 60.0, 59.9, 57.0, 56.8, 30.3, 27.4, 27.4,

26.8, 26.7. HRMS (ESI) m/z: Calculated for C₃₁H₃₅NNaO₅[M + Na]⁺

524.2407, found 524.2410.

4.3.48. Benzyl

(2S,3S)-2-(((benzyloxy)carbonyl)amino)-4-((tert-

butoxycarbonyl)amino)-3-hydroxybutanoate (24)

Benzyl bromide (2.45 g, 14.3 mmol) was added to a solution of 23

(4.80 g, 13.0 mmol) and DIPEA (2.45 mL, 14.3 mmol) in DMF (40 mL)

at 0 °C. The mixture was stirred for 48 h at room temperature and then

poured into ice-cold 1% aqueous HCl (150 mL). The obtained slurry

was supplemented with EtOAc (250 mL) and thoroughly shaken. The

organic layer was separated and the aqueous layer was extracted with

EtOAc (2 × 100 mL). The organic layers were combined, washed

successively with 5% aqueous NaHCO₃, water (50 mL), brine (50 mL),

and dried (Na₂SO₄). After evaporation of the solvent, the residue was

triturated with hexane (100 mL), stirred overnight, ﬁltered, and dried in

vacuo to give 24 as a white solid (5.69 g, 95%). [ ]²_D¹+ 0.4 (c 1.0,

MeOH). ¹H NMR (400 MHz, DMSO‑d₆) δ: 7.59 (d, J = 8.7 Hz, 1H),

7.40–7.25 (m, 10H), 6.64 (t, J = 6.6 Hz, 1H), 5.19 (d, J = 5.6 Hz, 1H),

5.12 (d, J = 13.5 Hz, 1H), 5.11 (d, J = 13.5 Hz, 1H), 5.06 (d,

J = 12.6 Hz, 1H), 5.03 (d, J = 12.6 Hz, 1H), 4.24 (dd, J = 8.7, 5.3 Hz,

1H), 3.88 (qui, J = 5.6 Hz, 1H), 3.10 (dt, J = 13.8, 5.5 Hz, 1H), 3.05

(dt, J = 13.8, 6.6 Hz, 1H), 1.36 (s, 9H). ¹³C NMR (101 MHz, DMSO‑d₆)

δ: 170.3, 156.1, 155.7, 136.8, 135.9, 128.3, 127.9, 127.8, 127.7, 127.6,

77.8, 69.8, 65.9, 65.7, 57.7, 43.0, 28.2. HRMS (ESI) m/z: Calculated for

4.3.46. (R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-

butoxy)pentanoic acid (21)

(R)-2-Amino-5-(tert-butoxy)pentanoic acid (20) was prepared from

19 (963 mg, 1.92 mmol) and 10% Pd on activated carbon (600 mg,

0.56 mmol) as described for 7a. Acid 20 was obtained as a white solid

(271 mg, 74%), which was used in the next step without additional

puriﬁcation. HRMS (ESI) m/z: Calculated for C₉H₂₀NO₃[M + H]⁺

190.1438, found 190.1439.

To a white suspension of compound 20 from above (271 mg,

1.43 mmol) and NaHCO₃(132 mg, 1.58 mmol) in water (20 mL) was

dropwise added a solution of FmocOSu (483 mg, 1.43 mmol) in dioxane

(20 mL) over 5 min. The obtained mixture was stirred at room tem-

perature for 18 h and acidiﬁed with 6 M HCl to pH 3. Volatiles were

removed in vacuo and the waxy residue (374 mg) was puriﬁed by

C

₂₄H₃₁N₂O₇[M + H]⁺459.2126, found 459.2131.

16

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Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

4.3.49. Benzyl

(2S,3S)-2-(((benzyloxy)carbonyl)amino)-4-((tert-

for enantiomeric 29.³¹Thus, a solution of Fmoc-D-Orn(Boc)–OH (28)

(2.27 g, 5.0 mmol), solid paraformaldehyde (0.300 g, 10.0 mmol) and

camphorsulfonic acid (0.10 g, 0.4 mmol) in MeCN (25 mL) was heated

by microwaves at 120 °C in a sealed microwave vial for 2 min. The

reaction mixture was cooled, concentrated in vacuo, and ﬁltered

through a short silica pad using 7:3 petroleum ether/EtOAc and 100%

EtOAc as eluents. Combined ﬁltrates were dried (Na₂SO₄), con-

centrated, and the obtained oil was dried in vacuo to give the desired

compound 29 as a white foam (1.70 g, 73%). [ ]²_D²−19.1 (c 1.1,

MeOH). ¹H NMR (400 MHz, CDCl₃) δ: 8.50 (br s, 1H), 7.84–7.66 (m,

2H), 7.64–7.46 (m, 2H), 7.44–7.18 (m, 4H), 5.33–4.85 (m, 2H),

4.80–4.06 (m, 4H), 3.54–3.13 (m, 2H), 2.33–1.88 (m, 2H), 1.87–1.54

(m, 2H), 1.43 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ: 155.2, 154.5,

144.0, 141.4, 127.8, 127.3, 127.2, 125.3, 125.0, 120.1, 80.7, 68.3,

67.9, 58.0, 47.3, 46.6, 28.5, 28.4. HRMS (ESI) m/z: Calculated for

butoxycarbonyl)amino)-3-methoxybutanoate (25)

Ag₂O (7.25 g, 31.3 mmol) and MeI (8.88 g, 62.6 mmol) were added

to a solution of compound 24 (2.87 g, 6.3 mmol) in MeCN (40 mL). The

reaction mixture was stirred at room temperature for 3 days, ﬁltered

through a pad of celite and ﬁltrate was concentrated in vacuo. The re-

sidue was dissolved in DCM (150 mL), the obtained solution was suc-

cessively washed with saturated aqueous Na₂S₂O₃solution (50 mL), 5%

aqueous NaHCO₃solution (2 × 50 mL), water (50 mL), brine (50 mL),

and dried (Na₂SO₄). After evaporation of the solvent, the residue was

dried in vacuo to give crude compound 25 (2.95 g), which in the next

step without further puriﬁcation. Analytically pure 25 was prepared by

column chromatography on silica gel (gradient elution from 0% MeOH

in CHCl₃to 2% MeOH in CHCl₃). [ ]²_D¹+ 1.3 (c 1.0, MeOH). ¹H NMR

(400 MHz, CD₃OD) δ: (rotamer mixture) 7.44 – 7.23 (m, 10H), 5.18 (s,

2H), 5.10 (d, J = 12.4 Hz, 1H), 5.08 (d, J = 12.4 Hz, 1H), 4.57 (d,

J = 5.2, 1H), 3.67 (q, J = 5.7, 1H), 3.36 (s, 3H), 3.26 (dd, J = 14.4,

5.7, 1H), 3.18 (dd, J = 14.4, 6.2, 1H), 1.43 (s, 9H). ¹³C NMR (101 MHz,

CD₃OD) δ: (rotamer mixture) 171.7, 158.6, 158.4, 138.1, 137.1, 130.3,

129.5, 129.5, 129.3, 129.0, 128.8, 81.4, 80.4, 68.1, 67.8, 58.5, 56.1,

41.2, 28.8. HRMS (ESI) m/z: Calculated for C₂₅H₃₃N₂O₇[M + H]⁺

473.2282, found 473.2292.

C

₂₆H₃₁N₂O₆[M + H]⁺467.2177. Found: 467.2180.

4.3.53. (R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-

(methylamino)pentanoic acid (30)

Compound 30 was prepared according to the literature procedure

for enantiomeric 30.³¹Accordingly, a mixture of compound 29 (1.70 g,

3.6 mmol), triethylsilane (3.0 mL, 22 mmol), triﬂuoroacetic acid

(1.7 mL, 21.9 mmol), and dichloroethane (2.7 mL) was stirred at 60 °C

for 18 h in a sealed vial. The reaction mixture was cooled, concentrated,

and dried in vacuo to give the crude 30 as a white foam (2.06 g, the-

oretical amount 1.34 g). The crude product was used in the next step

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃) δ: 11.38–10.29

(m, 2H), 8.70–8.08 (m, 2H), 7.72 (d, J = 7.4 Hz, 2H), 7.60–7.42 (m,

2H), 7.36 (t, J = 6.2 Hz, 2H), 7.29–7.20 (m, 2H, overlapped with

CDCl₃), 4.70–3.81 (m, 4H), 3.29–2.69 (m, 2H), 2.62 (s, 3H), 2.07–1.42

4.3.50. (2S,3S)-2-Amino-4-((tert-butoxycarbonyl)amino)-3-

methoxybutanoic acid (26)

A stream of hydrogen was passed through a mixture of crude

compound 25 from above (2.90 g) and 10% Pd on activated carbon

(0.65 g, 0.6 mmol) in MeOH (50 mL) for 1 h. The reaction mixture was

ﬁltered through a pad of celite, the ﬁltrate was concentrated in vacuo

and the residue was triturated with MeCN (25 mL). The precipitate was

ﬁltered and puriﬁed by reverse phase column chromatography (gra-

dient elution from 0% MeCN in water to 50% MeCN in water) to give 26

as a white solid (0.62 g, 41% in two steps from 24). [ ]²_D²−24.0 (c 0.5,

MeOH). ¹H NMR (400 MHz, DMSO‑d₆) δ: 7.49 (br s, 3H), 6.98 (t,

J = 5.6 Hz, 1H), 3.62–3.56 (m, 1H), 3.42 (d, J = 3.2 Hz, 1H), 3.27 (s,

3H), 3.25–3.98 (m, 2H), 1.37 (s, 9H). ¹H NMR (101 MHz, CD₃OD) δ:

3.84 (d, J = 3.4, 1H), 3.77 (td, J = 6.5, 3.4, 1H), 3.46 (s, 3H), 3.33 (dd,

(m, 3H, overlapped with water). LCMS ESI (m/z): 369.5 [M + H]⁺

.

4.3.54. (R)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-5-((tert-

butoxycarbonyl)(methyl)amino)pentanoic acid (31)

To a solution of the crude 30 from above (2.06 g) in 1:1 (v:v) water/

THF mixture (60 mL) was added solid NaHCO₃(0.74 g, 8.8 mmol) at

0–5 °C (crushed ice), followed by a solution of Boc₂O (0.77 g, 3.5 mmol)

in THF (20 mL). The reaction mixture was stirred at room temperature

for 18 h, then partially evaporated to remove THF, acidiﬁed with 0.1 M

aqueous HCl solution to pH 4, and extracted with EtOAc (3 × 100 mL).

The organic extracts were combined, washed with water, brine, dried

(Na₂SO₄), and the solvent was evaporated. The residue was puriﬁed by

column chromatography on silica gel (gradient elution from 15:1 DCM/

EtOH to 5:1 DCM/EtOH) to give 31 as a white foam (1.05 g, 62% in two

steps from 29). [ ]²_D²−1.0 (c 1.01, MeOH). ¹H NMR (400 MHz,

DMSO‑d₆) δ: 12.64 (br s, 1H), 7.89 (d, J = 7.4 Hz, 2H), 7.72 (d,

J = 7.4 Hz, 2H), 7.61 (d, J = 8.6 Hz, 1H), 7.42 (t, J = 7.4 Hz, 2H),

7.32 (t, J = 7.4 Hz, 2H), 4.36–4.17 (m, 3H), 4.03–3.88 (m, 1H),

3.27–3.06 (m, 2H), 2.74 (s, 3H), 1.75–1.61 (m, 1H), 1.61–1.43 (m, 3H),

1.37 (s, 9H). ¹³C NMR (101 MHz, DMSO‑d₆) δ: 173.9, 156.1, 154.8,

143.9, 143.8, 140.7, 128.9, 127.6, 127.0, 125.3, 121.4, 120.1, 78.3,

J = 14.3, 7.2 Hz, 1H), 3.25 (dd, J = 14.3, 5.8 Hz, 1H), 1.44 (s, 9H). ¹³

C

NMR (CD₃OD) δ: 171.1, 158.6, 80.4, 79.8, 58.3, 56.3, 40.9, 28.7. HRMS

(ESI) m/z: Calculated for C₁₀H₂₁N₂O₅[M + H]⁺249.1445, found

249.1448.

4.3.51. (2S,3S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-

((tert-butoxycarbonyl)amino)-3-methoxybutanoic acid (27)

Compound 27 was prepared from 26 (0.56 g, 2.3 mmol), solid

NaHCO₃(0.47 g, 5.6 mmol), and FmocOSu (0.76 g, 2.3 mmol) as de-

scribed for 8a. After evaporation of solvent, the obtained colorless oil

was dissolved in MeCN (5 mL) and after a while a formation of white

precipitate was observed. The precipitate was ﬁltered, rinsed with

MeCN (2 × 2 mL), and dried in vacuo over P₂O₅to give 27 as a white

solid (0.74 g, 69%). [ ]²_D¹+ 4.1 (c 1.0, MeOH). ¹H NMR (400 MHz,

DMSO‑d₆) δ: 12.80 (br s, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.76 (d,

J = 7.2 Hz, 1H), 7.73 (d, J = 7.2 Hz, 1H), 7.64 (d, J = 9.2 Hz, 1H),

7.42 (td, J = 7.5, 0.9 Hz, 2H), 7.32 (td, J = 7.4, 0.9 Hz, 2H), 6.76 (t,

J = 5.7 Hz, 1H), 4.39 (dd, J = 9.2, 4.2 Hz, 1H), 4.33–4.20 (m, 3H),

3.64 (dd, J = 10.4, 5.7 Hz, 1H), 3.30 (s, 3H), 3.21–3.01 (m, 2H), 1.38

(s, 9H). ¹³C NMR (101 MHz, DMSO‑d₆) δ: 171.5, 156.2, 155.7, 143.8,

143.7, 140.7, 140.7, 127.6, 127.1, 125.4, 125.3, 120.1, 79.4, 77.9,

65.9, 57.2, 54.1, 46.6, 28.2. HRMS (ESI) m/z: Calculated for C₂₅H₃₀N₂

65.6, 53.6, 47.4, 46.7, 33.5, 28.0, 23.8. HRMS (ESI) m/z: Calculated for

+

C

₂₆H₃₃N₂O₆[M + H]

469.2333, found 469.2324. Anal. Calcd for

C

₂₆H₃₂N₂O₆× 0.25 H₂O (0.95%): C 66.02, H 6.92, N 5.92. Found: C

65.94, H 6.85, N 5.89.

4.3.55. Ni(II) complex of schiﬀ base of (S)-N-(2-benzoylphenyl)-1-

benzylpyrrolidine-2-carboxamide (BPB) and glycine ((S)–32)

The complex (S)–32 was prepared according to the literature pro-

Na]⁺493.1945, found 493.1947. Anal. Calcd for

cedure.⁴⁵[ ]²_D²+ 2136 (c 0.1, CHCl₃), [ ]²_D¹+ 1857 (c 0.1, MeOH); ref

NaO₇[M

+

45

:

[ ]²_D⁰+ 1972 (c 0.1, CHCl₃), ref 45: [ ]²_D⁵+ 2006 (c 0.1, MeOH). ¹H

C₂₅H₃₀N₂O₇× 0.05 H₂O (0.2%): C 63.70, H 6.44, N 5.94. Found: C

63.69, H 6.40, N 5.92.

NMR (400 MHz, CDCl₃) δ: 8.29 (dd, J = 8.7, 1.2 Hz, 1H), 8.07 (d,

J = 7.5 Hz, 2H), 7.56 – 7.46 (m, 3H), 7.42 (t, J = 7.5 Hz, 2H), 7.33 –

7.28 (m, 1H), 7.20 (ddd, J = 8.7, 7.0, 1.7 Hz, 1H), 7.10 (d, J = 7.1 Hz,

1H), 7.01 – 6.95 (m, 1H), 6.80 (dd, J = 8.2, 1.7 Hz, 1H), 6.72–6.67 (m,

1H), 4.48 (d, J = 12.7 Hz, 1H), 3.77 (d, J = 20.0 Hz, 1H), 3.72 – 3.66

4.3.52. (R)-3-(((9H-Fluoren-9-yl)methoxy)carbonyl)-1-(tert-

butoxycarbonyl)-1,3-diazepane-4-carboxylic acid (29)

Compound 29 was prepared according to the literature procedure

17

E. Loza, et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

(m, 1H), 3.68 (d, J = 20.0 Hz, 1H), 3.67 (d, J = 12.7 Hz, 1H), 3.47 (dd,

J = 10.8, 5.5, 1H), 3.41–3.28 (m, 1H), 2.63 – 2.52 (m, 1H), 2.48 – 2.36

(m, 1H), 2.20 – 2.01 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ: 181.5,

177.5, 171.8, 142.6, 134.8, 133.4, 133.3, 132.4, 131.9, 129.9, 129.7,

129.5, 129.3, 129.1, 126.4, 125.8, 125.3, 124.4, 121.0, 70.0, 63.3,

61.4, 57.6, 30.9, 23.8. HRMS (ESI) m/z: Calculated for C₂₇H₂₆N₃NiO₃

[M + H]⁺498.1322, found 498.1337. Spectra were in agreement to

those reported in the literature.⁴⁶

(2.69 g, 41.4 mmol) in water (15 mL) was added. The mixture was

stirred at room temperature for 2 h, then diluted with DCM (400 mL),

washed with brine (3 × 70 mL), and dried (Na₂SO₄). Solvents were

evaporated and the residue was dissolved in allyl alcohol (80 mL) and

toluene (80 mL). The resulting mixture was stirred at 80 °C for 16 h,

then concentrated in vacuo to dryness and puriﬁed by two consecutive

column chromatographies on silica gel (eluent for the ﬁrst column

CHCl₃/acetone, 10:1, eluent for the second column CHCl₃/MeOH,

100:1) to give (S)(2S,3S)-36 (3.49 g, 66%, 95:5 d.r.). [ ]²_D²+ 2401 (c

0.1, CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ: 8.26 (d, J = 8.6 Hz, 1H),

8.02 (d, J = 7.6 Hz, 2H), 7.57 – 7.41 (m, 3H), 7.33 (t, J = 7.4 Hz, 2H),

7.26–7.21 (m, 1H), 7.20 – 7.11 (m, 2H), 6.93 – 6.84 (m, 1H), 6.67 –

6.59 (m, 2H), 5.86 (ddt, J = 17.2, 10.5, 5.4 Hz, 1H), 5.27 (dq,

J = 17.2, 1.5 Hz, 1H), 5.20 (dq, J = 10.5, 1.4 Hz, 1H), 5.09 (t,

J = 6.3 Hz, 1H), 4.44 (ddt, J = 13.4, 5.3, 1.4 Hz, 1H), 4.42 (d,

J = 12.6 Hz, 1H), 4.36 (ddt, J = 13.4, 5.5, 1.5 Hz, 1H), 4.06 (d,

J = 4.0 Hz, 1H), 3.62 (d, J = 12.6, 1H), 3.52–3.43 (m, 2H), 3.39–3.23

(m, 1H), 3.04 (ddd, J = 14.0, 6.7, 5.0 Hz, 1H), 2.91 (ddd, J = 14.0, 9.7,

5.9 Hz, 1H), 2.86–2.75 (m, 1H), 2.60 – 2.46 (m, 1H), 2.13–2.02 (m,

2H), 1.98 (d, J = 6.7, 3H), 1.96 – 1.80 (m, 1H). ¹³C NMR (101 MHz,

CDCl₃) δ: 180.2, 177.9, 171.2, 155.6, 142.5, 133.6, 133.5, 133.2, 132.9,

132.4, 131.5, 129.6, 129.0, 128.9, 128.8, 128.6, 127.9, 126.7, 126.1,

123.3, 120.6, 117.1, 71.4, 70.4, 65.1, 63.4, 56.7, 43.5, 37.3, 30.7, 23.0,

13.8. HRMS (ESI) m/z: Calculated for C₃₄H₃₇N₄NiO₅(M + H)⁺

639.2112, found 639.2116.

4.3.56. Ni(II) complex of Schiﬀ base of BPB and (2S,3S)-2-amino-5-

methoxy-3-methyl-5-oxopentanoate ((S)(2S,3S)–33)

Compound (S )(2S,3S)–33 was synthesized according to the litera-

ture procedure.³²Thus, to a solution of complex (S)–32 (6.22 g,

12.5 mmol) in DMF (25 mL) was added DBU (0.95 g, 6.24 mmol). The

obtained mixture was stirred at room temperature for 15 min and then

methyl crotonate (1.37 g, 13.7 mmol) was added dropwise. After stir-

ring at room temperature for 60 h, LCMS analysis of the crude reaction

showed the formation of diastereomeric products with 70:30 ratio. The

reaction mixture was poured into cold 5% aqueous acetic acid (250 mL)

and stirred to initiate crystallization. The precipitate was ﬁltered,

thoroughly washed with water, and dried in vacuo to aﬀord a red solid

(4.32 g). The solid was vigorously stirred with Et₂O (40 mL) overnight

at room temperature, ﬁltered, washed with Et₂O, and dried in vacuo to

give (S)(2S,3S)–33 as red crystals (3.94 g, 52%, 95:5 d.r.). [ ]²_D²+ 1918

(c 0.1, CHCl₃). ¹H NMR (400 MHz, DMSO‑d₆) δ: 8.35 (d, J = 7.5 Hz,

2H), 8.06 (d, J = 8.6, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.55 (t, J = 7.2 Hz,

1H), 7.51–7.44 (m, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.15–7.04 (m, 3H),

6.65 (t, J = 7.6 Hz, 1H), 6.49 (dd, J = 8.2, 1.3 Hz, 1H), 4.04 (d,

J = 12.3 Hz, 1H), 3.77 (d, J = 3.5 Hz, 1H), 3.61–3.51 (m, 2H), 3.37 (s,

3H), 3.31–3.24 (m, 1H, overlapped with water), 3.23–3.06 (m, 1H),

2.55–2.37 (m, 2H, overlapped with DMSO), 2.24–2.04 (m, 4H),

2.04–1.93 (m, 1H), 1.88 (d, J = 6.1 Hz, 3H). ¹³C NMR (101 MHz,

DMSO‑d₆) δ: 180.1, 175.6, 171.1, 170.7, 142.5, 134.8, 133.8, 132.9,

131.4, 129.5, 128.9, 128.7, 128.5, 128.2, 127.9, 127.1, 125.7, 122.9,

119.9, 72.4, 69.8, 62.9, 57.1, 51.2, 37.5, 33.9, 30.3, 22.8, 15.8. HRMS

(ESI) m/z: Calculated for C₃₂H₃₄N₃NiO₅[M + H]⁺598.1846, found

598.1855.

4.3.59. (2S,3S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-

(((allyloxy)carbonyl)amino)-3-methylbutanoic acid ((2S,3S)-37)

Concentrated aqueous HCl (3.5 mL, 42.1 mmol) was slowly added

to a stirred solution of complex (S)(2S,3S)-36 (3.37 g, 5.3 mmol) in

MeOH (53 mL) and the resulting mixture was heated at 60 °C for 4 h.

Volatiles were evaporated in vacuo to dryness, the residue was sus-

pended in water (40 mL), the insoluble solid was ﬁltered oﬀ and wa-

shed with water (3 × 10 mL). To the combined ﬁltrates was added solid

NaHCO₃(1.77 g, 21.1 mmol) and the resulting solution was washed

with CHCl₃(3 × 20 mL). To the aqueous layer a solution of FmocOSu

(2.67 g, 7.9 mmol) in dioxane (40 mL) was added, and the resulting

solution was stirred at room temperature for 64 h, whereupon it was

supplemented with water (20 mL) and EtOAc (40 mL), and acidiﬁed

with 6 M aqueous HCl to pH 3. The organic layer was separated, wa-

shed with 1 M aqueous KHSO₄solution (20 mL), water (20 mL), and

brine (20 mL). The solvent was evaporated and the residue was puriﬁed

by column chromatography on silica gel (eluted consecutively with

CHCl₃, then with 20:1 CHCl₃/MeOH, and, ﬁnally, with 100:5:1 CHCl₃/

MeOH/AcOH) to give (2S,3S)-37 as a white solid (1.17 g, 51%, 99%

d.e.) [ ]²_D²+ 13.8 (c 1.02, MeOH). ¹H NMR (400 MHz, DMSO‑d₆) δ:

12.68 (br s, 1H), 7.89 (d, J = 7.5, 2H), 7.74 (d, J = 7.5, 2H), 7.60 (d,

J = 8.7, 1H), 7.42 (td, J = 7.5, 1.0, 2H), 7.33 (td, J = 7.4, 1.0, 2H),

7.20 (t, J = 5.8, 1H), 5.90 (ddt, J = 17.2, 10.5, 5.4, 1H), 5.27 (dq,

J = 17.2, 1.6, 1H), 5.16 (dq, J = 10.5, 1.4, 1H), 4.46 (dt, J = 5.4, 1.5,

2H), 4.35 – 4.18 (m, 3H), 4.03 (dd, J = 8.7, 4.8, 1H), 3.10 – 2.94 (m,

2H), 2.19–2.02 (m, 1H), 0.87 (d, J = 6.9, 3H). ¹³C NMR (101 MHz,

DMSO‑d₆) δ: 172.8, 156.4, 156.2, 143.8, 143.8, 140.7, 140.7, 133.8,

127.7, 127.1, 125.3, 120.1, 116.9, 65.8, 64.3, 56.6, 46.7, 42.9, 35.3,

14.5. HRMS (ESI) m/z: Calculated for C₂₄H₂₇N₂O₆(M + H)⁺439.1864,

found 439.1864.

4.3.57. Ni(II) complex of Schiﬀ base of BPB and (2S,3S)-2-amino-4-

carboxy-3-methylbutanoate ((S)(2S,3S)-34)

1 M aqueous NaOH solution (57 mL) was added to a solution of (S)

(2S,3S)–33 (5.70 g, 9.5 mmol) in 3:1 (v:v) mixture of EtOH and water

(200 mL). The resulting solution was stirred for 1 h at room tempera-

ture, poured into cold 5% aqueous acetic acid (500 mL), and extracted

with CHCl₃(3 × 300 mL). The combined extracts were evaporated, and

the oily residue was mixed with water (100 mL). The solid material was

ﬁltered, thoroughly washed with water, and dried in vacuo to give (S)

(2S,3S)-34 as red crystals (4.89 g, 88%, 95:5 d.r.). [ ]²_D²+ 2498 (c 0.1,

CHCl₃). ¹H NMR (400 MHz, CDCl₃) δ: 12.5–9.5 (br s, 1H), 8.24 (d,

J = 8.6 Hz, 1H), 8.04 (d, J = 7.5 Hz, 2H), 7.64 – 7.41 (m, 3H), 7.34 (t,

J = 7.5 Hz, 2H), 7.29–7.10 (m, 3H), 6.93 (d, J = 7.3 Hz, 1H),

6.74–6.61 (m, 2H), 4.40 (d, J = 12.7 Hz, 1H), 4.13 (d, J = 3.7 Hz, 1H),

3.63 (d, J = 12.7 Hz, 1H), 3.55–3.42 (m, 2H), 3.40–3.23 (m, 1H),

2.88–2.72 (m, 1H), 2.65–2.46 (m, 1H), 2.33–2.16 (m, 2H), 2.16–1.90

(m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ: 180.5, 178.9, 173.7, 172.3,

142.6, 133.9, 133.5, 133.3, 132.8, 131.6, 130.3, 129.3, 129.2, 129.1,

129.0, 127.8, 126.7, 126.3, 123.6, 121.0, 72.6, 70.6, 63.7, 56.9, 39.2,

34.2, 30.8, 23.2, 16.5. HRMS (ESI) m/z: Calculated for C₃₁H₃₂N₃NiO₅

(M + H)⁺584.1690, found 584.1717.

4.3.60. (2S,3S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-

amino-3-methylbutanoic acid ((2S,3S)-38)

To a mixture of (2S,3S)-37 (1.96 g, 4.5 mmol), PdCl₂(PPh₃)₂

(66 mg, 0.09 mmol) and water (0.45 mL, 25.0 mmol) in DCM (90 mL)

was added n-Bu₃SnH (2.6 mL, 9.8 mmol) and the reaction mixture was

stirred under argon atmosphere at room temperature for 6 h. Solvent

were evaporated and the residue was triturated with Et₂O. The formed

white precipitate was ﬁltered and dried in vacuo to give (2S,3S)-38 as a

white solid (1.34 g, 84%). [ ]²_D²+ 12.2 (c 1.00, MeOH). ¹H NMR

4.3.58. Ni(II) complex of Schiﬀ base of BPB and (2S,3S)-4-(((allyloxy)

carbonyl)amino)-2-amino-3-methylbutanoate ((S)(2S,3S)-36)

To a suspension of (S)(2S,3S)-34 (4.84 g, 8.3 mmol) and Et₃N

(4.6 mL, 33.1 mmol) in THF (30 mL) at 0 °C was added ethyl chlor-

oformate (2.4 mL, 25 mmol). After stirring at room temperature for

30 min, the reaction mixture was cooled to 0 °C and a solution of NaN₃

18

E. Loza, et al.

Bioorganic&MedicinalChemistryxxx(xxxx)xxxx

(400 MHz, DMSO‑d₆) δ: 8.59 (br s, 3H), 7.89 (d, J = 7.5 Hz, 2H), 7.71 –

7.60 (m, 2H), 7.41 (t, J = 7.5 Hz, 2H), 7.37–7.30 (m, 2H), 6.57 (d,

J = 4.8, 1H), 4.32 – 4.19 (m, 3H), 3.78–3.72 (m, 1H), 2.95 (dd,

J = 12.7, 3.1 Hz, 1H), 2.88 (dd, J = 12.7, 4.8 Hz, 1H), 2.22–2.10 (m,

1H), 0.82 (d, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆+ drop of

TFA) δ: 169.5, 142.7, 139.6, 137.6, 135.4, 129.6, 129.0, 127.4, 124.0,

121.5, 121.3, 120.1, 109.7, 54.1, 48.9, 41.6, 32.9, 12.1. HRMS (ESI) m/

z: Calculated for C₂₀H₂₃N₂O₄(M + H)⁺355.1652, found 355.1655.

Author contributions

The manuscript was written through contributions of all authors. All

authors have given approval to the ﬁnal version of the manuscript.

Conﬂict of interest

M. G. is founder and shareholder of Nosopharm.

Declaration of Competing Interest

4.3.61. (2S,3S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-4-

((tert-butoxycarbonyl)amino)-3-methylbutanoic acid ((2S,3S)-39)

Solid NaHCO₃(0.38 g, 4.5 mmol) was added to a solution of

(2S,3S)-38 (0.80 g, 2.3 mmol) in a 1:1 (v:v) mixture of water and THF

(12 mL), followed by a solution of Boc₂O (0.99 g, 4.5 mmol) in THF

(5 mL). The resulting mixture was stirred at room temperature for 18 h

whereupon it was partially evaporated to remove THF, acidiﬁed with

6 M aqueous HCl to pH ~ 3 and extracted with EtOAc (3 × 30 mL).

Combined organic extracts were washed with 1 M KHSO₄solution

(1–5 mL), water (15 mL), brine (15 mL), dried (Na₂SO₄), and solvents

were evaporated. The obtained colorless oil was stirred with pentane

(20 mL) overnight and the precipitated solid was ﬁltered to give crude

(2S,3S)-39 (0.87 g). The purity of the product was increased by dis-

solving in Et₂O (5 mL) and precipitating with pentane. After ﬁltering

and drying in vacuo the desired compound (2S,3S)-39 was obtained as a

white solid (0.76 g, 74%). [ ]²_D²+ 10.7 (c 1.03, MeOH). ¹H NMR

(400 MHz, DMSO‑d₆) δ: 12.63 (br s, 1H), 7.89 (d, J = 7.5 Hz, 2H), 7.74

and 7.67–7.61 (d, J = 7.5 Hz and m, altogether 2H), 7.56 (d,

J = 8.8 Hz, 1H), 7.42 (td, J = 7.5, 0.9 Hz, 2H), 7.32 (td, J = 7.5,

1.0 Hz, 2H), 6.79 (t, J = 5.7 Hz, 1H), 4.32–4.19 (m, 3H), 4.01 and

3.99–3.90 (dd, J = 8.8, 4.6 Hz and m, altogether 1H), 2.98 (dt,

J = 13.5, 6.2 Hz, 1H), 2.91 (dt, J = 13.5, 6.9 Hz, 1H), 2.20–1.99 (m,

1H), 1.38 (s, 9H), 0.85 (d, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz,

DMSO‑d₆) δ: 172.8, 156.3, 155.9, 143.8, 140.7, 127.6, 127.0, 125.3,

120.1, 77.6, 65.8, 56.6, 46.6, 42.4, 35.3, 28.2, 14.7. HRMS (ESI) m/z:

Calculated for C₂₅H₃₀N₂NaO₆(M + Na)⁺477.1996, found 477.2000.

Anal. Calcd for C₂₅H₃₀N₂O₆× 0.15 H₂O (0.6%): C 65.67, H 6.68, N

6.13. Found: C 65.68, H 6.69, N 6.00.

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂu-

ence the work reported in this paper.

Acknowledgements

This work has received ﬁnancial support [as a part of European

Gram-negative Antibacterial Engine (ENABLE) project WP5b] from the

Innovative Medicines Initiative (IMI) Joint Undertaking under grant

agreement n115583, resources of which are composed of ﬁnancial

contribution from the European Union’s Seventh Framework Program

(FP7/20072013) and European Federation of Pharmaceutical Industry

Associations (EFPIA) companies’ in kind contribution. The ENABLE

project is also ﬁnancially supported by contributions from Academic

and SME partners (where SME stands for small and medium-sized en-

terprises). This work has also received ﬁnancial support from OSEO and

Region Languedoc-Roussillon under grant agreement A1010014J and

from DGA under grant agreement 122906117. We thank Prof. K.

Jaudzems for assistance with NMR experiments and Dr. A. Kinens for

docking studies. We also thank Dr. D. Lola and K. Kalinins for the

synthesis of building blocks.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.bmc.2020.115469.

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