Azetidinimines as a Novel Series of Non-Covalent Broad

doi.org/10.26434/chemrxiv.11897157.v1

Azetidinimines as a Novel Series of Non-Covalent Broad-SpectrumInhibitors of β-Lactamases with Submicromolar Activities AgainstCarbapenemases of Classes A, B and DEugénie Romero, Saoussen Oueslati, Mohamed Benchekroun, Agathe C. A. D’Hollander, Sandrine Ventre,Kamsana Vijayakumar, Corinne Minard, Cynthia Exilie, Linda Tlili, Pascal Retailleau, Agustin Zavala, EddyElisée, Edithe Selwa, Laetitia A. Nguyen, Alain Pruvost, Thierry Naas, Bogdan I. Iorga, Robert Dodd, KevinCariou

Submitted date: 25/02/2020 • Posted date: 25/02/2020Licence: CC BY-NC-ND 4.0Citation information: Romero, Eugénie; Oueslati, Saoussen; Benchekroun, Mohamed; D’Hollander, Agathe C.A.; Ventre, Sandrine; Vijayakumar, Kamsana; et al. (2020): Azetidinimines as a Novel Series of Non-CovalentBroad-Spectrum Inhibitors of β-Lactamases with Submicromolar Activities Against Carbapenemases ofClasses A, B and D. ChemRxiv. Preprint. https://doi.org/10.26434/chemrxiv.11897157.v1

The increasingly worrisome situation of antimicrobial resistances has pushed synthetic chemists to designoriginal molecules that can fight these resistances. To do so, inhibiting β-lactamases, one of the main modesof resistance to β-lactam antibiotics, is one of the most sought-after strategies, as recently evidenced by thedevelopment and approval of avibactam, relabactam and vaborbactam. Yet molecules able to inhibitsimultaneously β-lactamases belonging to different molecular classes remain scarce and currently there is nometallo-β-lactamase inhibitor approved for clinical use. Having recently developed a synthetic methodology toaccess imino-analogues of β-lactams (Chem. – Eur. J. 2017, 23, 12991,see ref) we decided to evaluate themas potential β-lactamase inhibitors and specifically against carbapenemases, which can hydrolyze andinactivate penicillins, cephalosporins and carbapenems. Herein we eport our findings that show that our newlydeveloped family of molecules are indeed excellent β-lactamase inhibitors and that our lead compound caninhibit NDM-1 (0.1 µM), KPC-2 (0.4 µM), and OXA-48 (0.6 µM) even though these three enzymes belong tothree different molecular classes of carbapenemases. This lead compound also inhibits the ESBL CTX-M-15and the cephalosporinase CMY-2, it is metabolically stable, and can repotentiate imipenem against a resistantstrain of Escherichia coli expressing NDM-1.

File list (2)

download fileview on ChemRxivmanuscriptAZETI.pdf (1.86 MiB)

http://doi.org/10.26434/chemrxiv.11897157.v1

https://chemrxiv.org/authors/Kevin_Cariou/6910475

https://chemrxiv.org/authors/Kevin_Cariou/6910475

https://chemrxiv.org/ndownloader/files/21818226

https://chemrxiv.org/articles/Azetidinimines_as_a_Novel_Series_of_Non-Covalent_Broad-Spectrum_Inhibitors_of_-Lactamases_with_Submicromolar_Activities_Against_Carbapenemases_of_Classes_A_B_and_D/11897157/1?file=21818226

download fileview on ChemRxivSupplementaryAZETI-ChemRxiv.pdf (4.49 MiB)



1

Azetidinimines as a novel series of non-covalent broad-spectrum

inhibitors of β-lactamases with submicromolar activities against

carbapenemases of classes A, B and D.

Eugénie Romero,†,# Saoussen Oueslati,‡,‖,# Mohamed Benchekroun,†,◊ Agathe C. A. D’Hollander,†,◊

Sandrine Ventre,† Kamsana Vijayakumar,† Corinne Minard,† Cynthia Exilie,‡,‖ Linda Tlili,‡,‖ Pascal

Retailleau,† Agustin Zavala,† Eddy Elisée,† Edithe Selwa,† Laetitia A. Nguyen,§ Alain Pruvost,§ Thierry

Naas, *,‡,‖,Ʇ,¶,◊ Bogdan I. Iorga,*,† Robert H. Dodd,† Kevin Cariou*,†,○

† Université Paris-Saclay, CNRS, Institut de Chimie des Substances Naturelles, LabEx LERMIT, UPR 2301, 91198, Gif-sur-Yvette,

France.

‡ UMR1184, Inserm, Université Paris-Saclay, LabEx LERMIT, Hôpital Bicêtre, Le Kremlin-Bicêtre, France.

‖ Bacteriology-Hygiene Unit, Hôpital Bicêtre, Le Kremlin-Bicêtre, France.

§ Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé, Gif-sur-Yvette, France.

Ʇ EERA Unit "Evolution and Ecology of Resistance to Antibiotics Unit, Institut Pasteur-AP-HP-Université Paris-Saclay, Paris,

France.

¶ Associated French National Reference Center for Antibiotic Resistance: Carbapenemase-Producing Enterobacteriaceae, Le

Kremlin-Bicêtre, France.

○ Current address: Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for

Inorganic Chemical Biology, 75005 Paris, France.

ABSTRACT

The rise of resistances in Gram negative bacteria is reaching an extremely worrying situation

and one of the main causes of resistance is the massive spread of very efficient β-lactamases,

which render most β-lactam antibiotics useless. Herein, we report the development of a series

of imino-analogs of β-lactams (namely azetidinimines) as efficient non-covalent inhibitors of

β-lactamases. Despite the structural and mechanistic differences between serine-β-

lactamases KPC-2 and OXA-48 and metallo-betalactamase NDM-1, all three enzymes can be

inhibited at a submicromolar level by compound 7dfm, which can also repotentiate imipenem

against a resistant strain of Escherichia coli expressing NDM-1. We show that this compound

2

can efficiently inhibit not only the three main clinically-relevant carbapenemases of Ambler

classes A, B and D, but also β-lactamases of all four classes (A, B, C and D). Our results pave

the way for the development of a new structurally original family of non-covalent broad-

spectrum inhibitors of β-lactamases.

TABLE OF CONTENTS

3

The discovery of penicillin was the start of a golden age for antibiotherapy that is now

threatened by exponentially increasing antibioresistance phenomena.1 Because β-lactams

(Figure 1a) have been, and still are, the most prescribed antibiotics worldwide, resistance

against them is particularly alarming. Gram negative bacteria (GNB), the main mechanism of

β-lactam resistance is due to the production of ß-lactamases, enzymes capable of hydrolyzing

β-lactams. Even carbapenems, the most powerful β-lactams, are not spared by

metallo-β-lactamases (MBLs) such as NDM-1, IMP-1 or VIM-1 (class B) and/or by some

clinically worrisome serine β-lactamases (SBLs) such as KPC-2 or OXA-48 (classes A and D,

respectively).2,3 The discovery of novel antibiotics acting on novel targets is difficult to foresee

and strategies to overcome β-lactam resistance4,5 especially via enzyme inhibition6,7 may

preserve our current therapeutic arsenal. This strategy was implemented more than 30 years

ago with the development of β-lactamase inhibitors (BLIs) including clavulanic acid or

sulbactam (Figure 1b), but was not actively pursued until recently. Since 2012 several new

broad-spectrum inhibitors of class A and class C β-lactamases have emerged. Of significant

interest, avibactam8,9 and vaborbactam10 (Figure 1b) were approved by the FDA for clinical use

whilst many of their congeners, in particular diazabicyclooctanes,11–15 are currently going

through preclinical or clinical development. These compounds are mainly able to efficiently

inhibit SBLs, including carbapenemases, of class A, C and sometimes D, but generally do not

inhibit MBLs (class B). In parallel, continuous efforts for the development of efficient MBL

inhibitors16 have recently led to the identification of promising molecules that can bind to zinc

atoms of the active site of MBLs, such as thiols17 – including the clinically available

antihypertensive agent L-captopril18 –, aspergillomarasmine A,19 rhodanines20 and their

thienolate derivatives21 or heteroaryl-carboxylic acids such as ANT43122 (Figure 1c). Yet there is

still no MBL inhibitor available for clinical use. Moreover, the emergence of bacterial isolates

4

producing two or even three different carbapenemases of different classes now dictates the

development of inhibitors capable of simultaneously inhibiting SBLs and MBLs. So far, apart

from some polyphenolic derivatives with moderate activities,23 only boronic acids have been

reported to efficiently inhibit both SBLs and MBLs (Figure 1d).

Figure 1. Representative structures of (a) β-lactam antibiotics (in bold: year of entry to the

market; in italic: year of resistance appearance); (b) serine-β-lactamase inhibitors (in bold: year

of entry to the market); (c) metallo-β-lactamase inhibitors; (d) dual serine-/metallo-β-lactamase

inhibitors.

5

Rigid cyclic analogues of vaborbactam such as VNRX-5133 (currently in phase 3 clinical trials)

were the first molecules shown to inhibit all classes of β-lactamases (Figure 1d).24–27 VNRX-5133

exhibits submicromolar inhibitory activity against most MBLs and SBLs, being only slightly less

active against IMP-1 (class B) and OXA-48 (class D). Very recently, thiol/boronic acid hybrids

(such as MS18) were developed and demonstrated interesting dual inhibition properties against

SBLs and MBLs.28 MS18 and its congeners also possess a rather broad scope but show limited

effects against NDM-1 (class B) and OXA-48.

In this context, we sought to develop novel inhibitors that could block the activities of both

SBLs and MBLs with comparable efficiency, specifically targeting the three most clinically-

relevant carbapenemases: KPC-2 (class A), NDM-1 (class B) and OXA-48 (class D).

We addressed this challenge by exploring uncharted chemical space around the β-lactam

nucleus. Synthetically, this four-membered ring can be obtained by a [2+2] cycloaddition

between a ketene and an imine,29 a reaction named the Staudinger synthesis after its

discoverer (Scheme 1a).30 We recently reported that carefully substituted ynamides31,32 can

be used as precursors for the in situ generation of ketenimines33,34 under mild conditions,

which can be intercepted by various heterocyclic nucleophiles35 or can undergo a microwave

assisted [2+2] cycloaddition with imines.36 By replacing the ketene (C=C=O) with the in situ

generated iminoketene (C=C=NR), the reaction directly led to an azetidinimine (Scheme 1b).

Such imino-β-lactams have only been rarely studied with respect to their synthesis37,38 and

never with respect to their biological and particularly, antibiotic activities. Taking into account

their structural similarities with β-lactams, we thus decided to evaluate their therapeutic

potential against carbapenemases.

6

Scheme 1. (a) Staudinger synthesis to access β-lactams; (b) imino-Staudinger synthesis to access

azetidinimines.

RESULTS AND DISCUSSION

Chemistry. Taking into consideration the scope and the fact that only aryl groups can be easily

incorporated in our previously developed methodology,36 we devised a convergent synthetic

plan to access as many structural variations as possible on the azetidinimine scaffold. The Ar1

group originated from aniline 1x, which was protected by a Boc group to give 2x (Scheme 2).

Coupling of 2x with brominated triisopropylsilylacetylene provided ynamide 3x after TBAF-

promoted desilylation. Condensation of benzaldehyde 4y with aniline 5z afforded imines 6yz.

Scheme 2. General synthetic plan to prepare tri-arylated azetidinimines 7xyz.

The key [2+2] cycloaddition was performed under microwave heating and gave azetidinimines

7xyz with concomitant loss of the Boc protecting group. For para-methoxy derivatives 7aaa

7

and 7aba, as well as para-benzyloxy derivative 7dfc, their corresponding para-phenols 7aam,

7abm and 7dfm were obtained by ether cleavage using BBr3 (for OMe) or AlCl3 (for OBn). More

than forty compounds were prepared using this route.

First results. The first compound evaluated was 7aaa, bearing a p-anisyl group on the

endocyclic nitrogen. This aryl group was initially chosen for synthetic reasons as it would

electronically favor the reaction and could also be easily derivatized. Initial assessment

showed that in the presence of 10 µM of 7aaa, the hydrolysis of imipenem by NDM-1 was

inhibited by 90% with an IC50 estimated to be between 2 and 5 µM (Table 1). This very

encouraging initial result prompted us to try and rationalize this inhibitory activity by

performing in silico molecular modeling studies (Figure 2). It appeared that the azetidinimine

does not behave like a β-lactam, whose carbonyl group is chelated by the zinc ions during the

hydrolysis process. Here, the methoxy group is coordinated by both zinc ions in the active site

and the four-membered ring acts as a scaffold to position the two phenyl rings in hydrophobic

regions of the enzyme active site. The Ar1 phenyl substituent is surrounded by Ser251, Asp212,

Ala 215 and Ser217 for both enantiomers, whereas the Ar2 phenyl substituent is positioned

near Asn220 in the R enantiomer and in the proximity of Val73, Ile 35, Met67 and Phe70 in

the S enantiomer (Figure 2). The two enantiomers of 7aaa were separated by chiral

chromatography and then tested separately, both showing similar inhibition of NDM-1.

8

Figure 2. Docking of compound 7aaa in the active site of NDM-1 (left) and schematic drawing

of its key interactions with the zinc ions (right). The enantiomers R and S of 7aaa are colored

in pink and green, respectively, and the zinc ions are colored in purple.

9

SAR for enzyme inhibitory activities. A systematic structure-activity relationship was then

undertaken by varying the aromatic groups around the azetidinimine nucleus and targeting

three carbapenemases: NDM-1, OXA-48 and KPC-2. In addition to its NDM-1 inhibitory

activity, 7aaa was found to be moderately active against KPC-2 but did not have any activity

against OXA-48 (Table 1). Variations of the Ar3 ring showed that minimal modifications such

as replacing the methoxy of 7aaa by an ethoxy (7aab) or a benzyloxy (7aac) led to an

improvement of the activity against all three enzymes, 7aac in particular displaying a sub-

micromolar activity against NDM-1 (0.4 µM). Similar activities to 7aab and 7aac were observed

when the methoxy group was in the meta position (7aad). However, the presence of two

methoxy groups (7aaf), or a benzo[d][1,3]dioxole moiety (7aag), led to a slight decrease of

efficiency against NDM-1 and a loss of activity for OXA-48 and KPC-2. This was partially

restored by incorporation of a 2,3-dihydrobenzo[b][1,4]dioxane, as 7aah was found to be

quite similar to 7aad in terms of inhibition profile. While 3,4,5-trimethoxy derivative 7aai only

maintained a low activity for NDM-1, aniline-derived compound 7aaj was found to inhibit all

three enzymes with IC50’s of 10.0, 7.0 and 3.5 µM, respectively. The incorporation of an iodine

atom (7aak) on the para position led to solubility issues preventing data from being obtained

against OXA-48 and KPC-2 although the anti NDM-1 activity could nevertheless be evaluated

to be in the 4.0-5.0 µM range. The thiomethyl derivative 7aal was also poorly soluble in water,

but its strong affinity for the active site Zn ions led to a high inhibition of NDM-1. Finally, the

free phenol 7aam was moderately active against KPC-2 and OXA-48 but again exhibited high

inhibitory properties against NDM-1 (0.8 µM). To conclude in this series of Ar3 variations, the

best pan-carbapenemase inhibitors were p-oxyphenyl compounds and especially those

bearing a benzyloxy (7aac) and a free hydroxy (7aam).

10

Table 1. Influence of Ar3 on NDM-1, OXA-48 and KPC-2 inhibitory activities compared to 7aaa.

IC50 (µM) or % inhibition

Compound NDM-1 OXA-48 KPC-2

2.0–5.0 N.E. 55% inhibition

at 10 µM

2.0–5.0 45% inhibition

at 10 µM 2.0–5.0

0.4 31% inhibition

at 10 µM 1.6

1.3 28% inhibition

at 10 µM 5.0–10.0


at 10 µM

17% inhibition at 10 µM

5.0 20% inhibition

at 10 µM


1.6 37% inhibition

at 10 µM 5.0–10.0


N.E. N.E.

10.0 7.0 3.5

4.0–5 N.D. N.D.

<1.0 15% inhibition

at 10 µM


0.8 26% inhibition

at 10 µM


N.E.: no effect (at 10 µM); N. D.: not determined

11

Keeping a para-methoxyphenyl group as Ar3, variations of Ar2 were then examined for activity

and compared to 7aaa (Table 2). The p-chloro analogue (7aba) maintained a similar activity

against NDM-1 and though some anti-OXA-48 activity was witnessed, its anti-KPC-2 effect was

largely lost. Both p-methoxy and p-fluoro analogues 7aca and 7ada were rather inefficient on

all three enzymes while the p-iodo, 3,5-dichloro and 2-naphthyl derivatives (7aea, 7aga and

7aha respectively) presented an interesting profile, being almost equipotent against NDM-1

and KPC-2 but somewhat less effective against OXA-48. In contrast, pyridinyl compound 7aia

was almost inactive on all three enzymes.

Table 2. Influence of Ar2 on NDM-1, OXA-48 and KPC-2 inhibitory activities compared to 7aaa.



2.0–5.0 N.E. 55% inhibition

at 10 µM


at 50 µM 44% inhibition

at 50 µM





N.D. 10% inhibition

at 10 µM

1.1 33% inhibition

at 10 µM 1.1


at 5 µM 2.0–5.0

2.0–5.0 10.0-20.0 5.0-7.0


N.E. 12% inhibition

at 100 µM


12

From these results, two Ar2 groups were selected: para-chloro and 2-naphthyl (as in 7aba and

7aha). At this stage, we wished to evaluate the influence of Ar1in both series (4-chloro in Table

3 and 2-naphthyl in Table 4). It is worth noting that 4-chloro derivatives generally exhibit a

greater ease of synthesis due to the superior reactivity of imine 6bz (or 6fz) in the [2+2]

cycloaddition. Compared to 7aba, the introduction of a substituent at the para position of Ar1

– whether a chloro (7bba), a bromo (7cba), an iodo (7dba), a trifluoromethyl (7eba) and to a

lesser extent a methoxy group (7fba) – was highly beneficial for the anti-NDM-1 activity, with

all compounds possessing IC50 values in the 0.5-0.8 µM range (Table 3).

Table 3. Influence of Ar1 on NDM-1, OXA-48 and KPC-2 inhibitory activities compared to 7aba and 7cfa.




at 50 µM 44% inhibition

at 50 µM

0.5


2.9

0.6 7.6 2.4

0.7 7.5 2.7

0.7


3.6

1.9


N.E.

1.2 46% inhibition

at 10 µM


0.5 8.5 2.9

0.8 5.9 8.7

N.E.: no effect (at 10 µM)

13

The IC50’s against KPC-2 were also improved with values between 2.0 and 3.6 µM, except for

(7fba). Additionally, the IC50’s against OXA-48 dropped under the 10 µM threshold for two

compounds (7cba and 7dba). A similar trend could be observed with an additional chloro at

the 2 position of Ar2 (2,4-dichloro series): the para-iodo (7dfa) and para-trifluoromethyl (7efa)

were found to be the most potent pan-inhibitors of carbapenemases NDM-1, OXA-48 and KPC-

2 in this series.

Table 4. Influence of Ar1 on NDM-1, OXA-48 and KPC-2 inhibitory activities compared to 7aha and 7ahb.



2.0–5.0 10.0-20.0 5.0-7.0

1.6


5.7

1.1 3.6 7.5

0.5 7.6 2.3

5.0–7.0 N.D. N.D.

0.6


2.8

0.5 6.2 2.5

0.6 7.4 2.4

N. D.: not determined

14

Similarly, excellent anti-NDM-1 activities were observed in the Ar2 = naphthyl series (Table 4),

with four compounds (7dha, 7bhb, 7dhb and 7fhb) having submicromolar IC50‘s. Activities

against KPC-2 and OXA-48 were also significantly improved, being always under 10 µM except

in the case of para-chlorinated derivatives 7bha and 7bhb (for OXA-48). Despite their high

activity, the increased lipophilicity brought by the naphthyl group (for 7dhb logPtheor = 6.94)

caused products in this series to be very poorly soluble and to easily form aggregates in the

assay media. Thus further studies within this series were stopped.

Having witnessed the highly favorable effect of the para substitution on Ar1 and keeping in

mind the importance of the para-alkoxy moiety on Ar3, further study of the influence of Ar2

was carried out with 7dba as reference (Table 5). All compounds in this series offered a rather

broad-spectrum of activity with most of them inhibiting all carbapenemases with an IC50 below

10 µM and even submicromolar for NDM-1. In contrast with the other compounds, p-fluoro

(7dda), p-iodo (7dea) and 3,5-dichloro (7dga) analogues were more active against OXA-48

than against KPC-2. Finally, keeping Ar1 = p-iodophenyl and Ar2 = 2,4-dichlorophenyl (7dfa),

variations on Ar3 were then further investigated. Surprisingly (compared with 7aaa and 7aac

in Table 1), the p-benzyloxyphenyl derivative (7dfc) proved to be less active than 7dfa (Table

5). 7dfc was then converted into a free phenol (7dfm) by AlCl3-mediated cleavage of the ether

bond (see Scheme 3). This compound exhibited submicromolar IC50 activity against the three

enzymes: 0.1 µM for NDM-1 (which could be explained by stronger interactions of the OH

function with the zinc ions of the active site), 0.4 µM for OXA-48 and 0.6 µM for KPC-2. While

the homologous benzylic alcohol analogue 7dfn was found to be less active, the corresponding

carboxylic acid 7dfo (obtained by oxidation of 7dfn with KMnO4, see Scheme 3) was the most

active compound on NDM-1 so far (0.07 µM) but with a complete loss of activity against OXA-

48.

15

Table 5. Influence of Ar2 compared to 7dba and of Ar3compared to 7dfa on NDM-1, OXA-48 and KPC-2 inhibitory activities

IC50 (µM)


0.7 7.5 2.7

1.0 4.2 9.5

0.7 4.4 8.5

0.5 7.6 2.3

4.5 4.8 42% inhibition

at 10 µM

0.5 8.5 2.9

0.8 41% inhibition

at 10 µM 5.5

0.1 0.4 0.6

3.0 N.E. 6.0

0.07 N.E. 6.0


16

Scheme 3. Synthesis of compounds 7dfn and 7dfo.

Complementary assays. Having found a lead (7dfm) in our novel azetidinimine BLI series, we

then performed complementary assays to ascertain its therapeutic potential. First, it was

screened at 10 µM against a wider panel of BLs (Table 6). At this concentration a complete

inhibition of three NDM variants (NDM-4, NDM-7 and NDM-9) and of VIM-1 (Class B) was

observed. In contrast, VIM-52 (class B) was not affected. The extended-spectrum β-lactamase

CTX-M-15 (class A) and the cephalosporinase CMY-2 (class C) were also inhibited (83% and

86%, at 10 µM, respectively). These latter results demonstrate that compound 7dfm can

inhibit not only carbapenemases from 3 classes but, more generally, BLs from all four classes.

Table 6. Additional enzymatic inhibitory activities (% at 10 µM) for 7dfm

Compound NDM-4 NDM-7 NDM-9 VIM-1 VIM-52 CTX-M-15 CMY-2

7dfm 100% 100% 100% 100% 0% 83% 86%

The metabolic stability of 7dfm was evaluated and the compound was found to possess an

excellent stability profile with a Clint of µL/min/mg protein in mouse hepatic cells (Table 7).

The toxicity of 7dfm was measured against both normal cells (MRC-5) and cancer cells (HCT-

116) and was found to be in the 20-30 µM range, that is, almost two orders of magnitude

higher than its IC50 against carbapenemases. Finally, compound 7dfm was evaluated for the

repotentiation of imipenem against the clinical strain of E. coli that expresses NDM-1 (amongst

other genes of resistance).39 In the absence of inhibitor the MIC was above the resistance

17

threshold at 16 µg/mL, in the presence of 20 µM of 7dfm it was divided by 2, while using 50

µM of 7dfm it was divided by 4, bringing it down to the intermediate/resistant limit.

Table 7. Metabolic stability, cytotoxicity and imipenem repotentiation for 7dfm

Stability IC50 (µM)c MIC (µg/mL)d

Non-

NADPHa CLintb MRC-5 HCT-116 0µM 20µM 50µM

7dfm 96% 13.9 19.9 32.3 16 8 4

a Stability after 45 min in the presence of mouse hepatic microsome in the absence of NADPH; b CLint was determined using mouse hepatic microsomes and is given in µL/min/mg protein; c inhibition of cell proliferation; d Minimum inhibitory concentration for imipenem using E. coli GUE-NDM139 clinical strain.

Molecular modeling Compound 7dfm was selected for a more in depth study of the

interaction with the three main clinically-relevant carbapenemases, NDM-1, KPC-2 and

OXA-48. The docking of these three enzymes with the two enantiomers of 7dfm was

performed using GOLD40 and the results are presented in Figure 4.

Figure 4: Docking conformations of the enantiomers R (a, b, c) and S (d, e, f) of compound 7dfm, in the active site

of NDM-1 (class B) represented as gray surface (a, d), KPC-2 (class A) represented as light blue surface (b, e) and

OXA-48 (class D) represented as pink surface (c, f). For NDM-1, the zinc ions are colored in purple, for KPC-2 the

residues Ser70, Lys73 and Ser130 are colored in yellow, purple and brown, respectively, and for OXA-48 the

residues Ser70 and Thr104 are colored in yellow and olive, respectively. Hydrogen bonds are represented as

springs colored in orange.

18

With NDM-1, both enantiomers of 7dfm interact in a similar manner as 7aaa (see Figure 2).

The phenol moiety is coordinated with the two zinc ions and the 4-iodo-phenyl substituent is

positioned in the same subpocket in both cases, possibly establishing a stabilizing halogen

bond with the side chain of Asp212. The 2,4-dichloro-phenyl substituent is positioned in a

hydrophobic environment bordered by residues Val73, Ile35, Met67 and Phe70 for the S

enantiomer (Figure 4d) and in the vicinity of Asn220 for the R enantiomer (Figure 4a).

The docking pose of KPC-2 with the R enantiomer (Figure 4b) shows two hydrogen bonds

between the phenol group of 7dfm and the side chains of Ser70 and Lys73. The 4-iodo-phenyl

substituent is positioned in a subpocket formed by the side chains of Asn218, His219 and

Glu276 and may form a halogen bond with the side chain of Asn218, whereas the 2,4-dichloro-

phenyl substituent is more solvent exposed. The S enantiomer makes hydrogen bonds with

the side chains of Ser70 and Ser130 through the phenol substituent (Figure 4e), and the

positions of the other two substituents are inverted compared with the R enantiomer.

The interaction of OXA-48 with the R enantiomer of 7dfm (Figure 4c) shows a hydrogen bond

between the phenol group and the side chain of Thr104, at the upper extremity of the binding

site, whereas the two other substituents are positioned more deeply in the binding site

groove. The S enantiomer establishes the same hydrogen bond between the phenol group

and the side chain of Thr104, with the positions of the 4-iodo-phenyl and 2,4-dichloro-phenyl

substituents inverted.

Conclusions

The evaluation of azetidinimines, imino-analogues of β-lactams, as β-lactamase inhibitors led

to the development of a new family of non-covalent carbapenemase inhibitors. Structural

19

optimization led to the identification of phenol 7dfm as the lead compound, which can

strongly inhibit MBL NDM-1 (0.1 µM), class A SBL KPC-2 (0.4 µM), and class D SBL OXA-48 (0.6

µM). Molecular modeling showed that this compound does not mimic a β-lactam within the

enzyme active sites and that both enantiomers can interact with the enzymes. Provided one

key interaction (such as complexation with the zinc ions in NDM-1 or hydrogen bonds in KPC-

2 and OXA-48) is established, the rigid and compact structure of the central four-membered

ring indeed allows the other aryl groups to be allocated almost interchangeably in the enzyme

pocket to increase the affinity. Further refinement of the structure of these molecules will be

pursued to improve the efficiency of these β-lactamase pan inhibitors. The metabolic stability

of the hit compound and its efficient repotentiation of imipenem against a resistant E. coli

strain will serve as the basis for future in vivo studies.

Materials and Methods

General Procedure for Azetidinimine Synthesis. The imine (1.0 equiv.), the ynamide (2.0

equiv.) and the additive – silica gel (1.0 equiv.) or ZnOTf2 (10 mol%) – were successively added

in a microwave sealable tube and placed under argon before the addition of t-BuOLi – solid or

2.2 M in solution in THF – (2.0 equiv.) followed by extra dry DMF (0.3M). The sealed tube was

placed in a microwave apparatus for 1 h at 100 °C. The crude material was purified by flash

chromatography on silica gel or with preparative TLC. See Supporting Information for detailed

synthetic procedures and characterizations of the compounds.

In vitro β-lactamase inhibition assay. IC50 values were determined against the panel of

purified carbapenemases: OXA-48, KPC-2 and NDM-1 by spectrophotometric assay, using

ULTROSPEC 2000 UV spectrophotometer and the SWIFT II software (GE Healthcare, Velizy-

20

Villacoublay, France). Compounds were dissolved in DMSO stock solutions at 10 mM; more

dilute stocks were subsequently prepared as necessary by dissolving them also in DMSO. Assay

conditions were as follows: 100 mM phosphate buffer, pH 7 (supplemented with 50 μM Zn2+

when testing NDM-1, and with 50 mM NaHCO3 when testing OXA-48), 100 μM imipenem

(Sigma-aldrich, Saint-Quentin Fallavier, France). The reaction was monitored at 297 nm, time

course 600 seconds at 25°C with 3 min of incubation (compound/carbapenemase). Each

inhibitor compound was assayed at seven different concentrations, in triplicate for calculating

an error value with 95% confidence interval (ρ < 0.05). IC50 values were determined using the

equation IC50 = ((1/0.5 x v0) − m)/q, where v0 is the rate of hydrolysis of the reporter substrate

(v0 being the rate measured in the absence of inhibitor), q the y axis intercept and m the slope

of the resulting linear regression. Percentage of inhibition were obtained with a concentration

of 10 μM of compound.

Minimal Inhibition concentrations. MIC values were determined by broth microdilution, in

triplicate, in cation-adjusted Mueller Hinton broth according to the Clinical Laboratory

Standards Institute (CLSI, https://clsi.org/) guidelines. The enterobacterial clinical strain E. coli

NDM-1 GUE expressing the carbapenemase NDM-1 was used.39 Experiments were performed

in microtiter plates containing the medium with imipenem and inhibitors (dissolved in DMSO).

Three inhibitor concentrations were tested: 50, 100 and 200 μM. Plates were incubated

overnight at 37°C for 18−24 h.

Incubations in hepatic microsomes. Compounds (5 μM) were incubated in 0.5 mg/mL of

pooled male mouse liver microsomes (from Biopredic, France), in 0.1 M phosphate buffer at

pH 7.4 at 37 °C. After prewarming the mixture for 5 min, reactions were initiated by the

addition of NADPH (1 mM). Incubations (400 µL) were performed at 37 °C for 0, 5, 15, 30 and

21

45 min in duplicate and the reaction immediately terminated by adding 200 μL of cold

acetonitrile. Samples (including the control to evaluate non NADPH-dependent stability) were

centrifuged and the supernatant fractions analyzed by UPLC-MS/MS with multiple reaction

monitoring (MRM). Diphenhydramine was used as positive control in mouse liver microsomes

test. The MRM area response of the analyte was set to 100% with the T0 incubation, the

relative decrease in MRM area ratio intensity over time against that of the control (percent

parent decrease) was used to determine the half-life (t1/2) of compounds in the incubation.

Half-life values were calculated from the relationship:

T1/2 (min) = 0.693/k, where k is the slope of the Ln concentration vs time curve. The intrinsic

clearance (CLint) was calculated as: CLint = (0.693 x incubation volume (μL))/(t (min) x mg of

microsomal protein).

Cell culture and proliferation assay. Assays were carried out at the Institut de Chimie des

Substances Naturelles by the CIBI screening platform. Cell lines were obtained from the

American type Culture Collection (Rockville, USA) and were cultured according to the

supplier’s instructions. Briefly, human MRC-5 cells were grown in DMEM supplemented with

10% fetal calf serum (FCS) and 1% glutamine and HCT116 colorectal carcinoma cells were

grown in RPMI 1640 containing 10% FCS and 1% glutamine. All cell lines were maintained at

37 °C in a humidified atmosphere containing 5% CO2. Cell growth inhibition was determined

by an MTS assay according to the manufacturer’s instructions (Promega, Madison, WI, USA).

Briefly, the cells were seeded in 96-well plates (2.5 × 103 cells/well) containing 200 μL of

growth medium. After 24 h of culture, the cells were treated with the test compounds at

different final concentrations. After 72 h of incubation, 40 μL of resazurin was added for 2 h

before recording absorbance at 490 nm with a spectrophotometric plate reader. The IC50 value

22

corresponded to the concentration of compound inducing a decrease of 50% in absorbance

of drug-treated cells compared with untreated cells. Experiments were performed in

triplicate. Paclitaxel was used as the reference compound.

PCR, Cloning, Expression, and DNA Sequencing. Whole-cell DNA of the enterobacterales

expressing β-lactamases NDM-1, NDM-4, NDM-7, NDM-9, VIM-1, VIM-52, CTX-M-15, CMY-2

and OXA-48 was extracted, using the QIAamp DNA mini kit (Qiagen, Courtaboeuf, France) and

used as a template to amplify all the different genes. The sequences without the peptide signal

(predicted by SignalIP 4.1 Server, http://www.cbs.dtu.dk/services/SignalP-4.1/) encoding for

the mature protein, were obtained by PCR amplification, using the forward primers, which

included an NdeI restriction site, and the reverse primer which included an XhoI restriction

site and a deletion of the stop codon of the gene to allow the expression of an C_Term His tag.

Then, PCR product was cloned into pET41b vector (Invitrogen®, Life Technologies, Cergy-

Pontoise, France), using NdeI and XhoI restriction enzymes, to obtain a C-Term His8-tag. The

accuracy of the recombinant plasmid was verified by sequencing, using a T7 promoter and T7

terminator with an ABI Prism 3100 automated sequencer (Applied Biosystems, Thermo Fisher

Scientific, Les Ulis, France). The nucleotide sequences were analyzed by using software

available at the National Center for Biotechnology Information website

(http://www.ncbi.nlm.nih.gov).

Protein purification. An overnight culture of E. coli BL21 DE3 harboring recombinant pET41b

plasmids was used to inoculate 2 L of LB medium broth containing 50mg/L kanamycin. Bacteria

were cultured at 37 °C until an OD of 0.6 at 600 nm was reached. The expression of the β-

lactamase genes was carried out overnight at 22 °C with 0.2 mM IPTG as inducer. Cultures

were centrifuged at 6000 g for 15 min and then the pellets were resuspended with the binding

23

buffer (10 mM imidazole, 25 mM sodium phosphate pH 7.4 and 300 mM NaCl). Bacterial cells

were disrupted by sonication and the bacterial pellet was removed by two consecutive

centrifugation steps at 10000g for 1h at 4 °C; the supernatant was then centrifuged at 96000g

for 1h at 4 C. The soluble fractions were filtered and then passed through a HisTrapTM HP

column (GE Healthcare) and proteins were eluted with the elution buffer (500 mM imidazole,

25 mM sodium phosphate pH 7.4 and 300 mM NaCl). Finally, a gel filtration step was

performed with 100 mM sodium phosphate buffer pH 7 and 150 mM NaCl with a Superdex 75

column (GE Healthcare). The protein purity was estimated by SDS–PAGE. The pooled fractions

were dialyzed against 10 mM Tris-HCl pH 7.6, for NDM and VIM 50 µM of ZnSO4 was added,

then concentrated using Vivaspin columns (Sartorius, Aubagne, France). The concentrations

were determined by measuring the OD at 280 nm and with the extinction coefficients

obtained from the ProtParam tool (Swiss Institute of Bioinformatics online resource portal).41

Molecular modeling. The three-dimensional structure of compound 7aaa36 was retrieved

from the Cambridge Structural Database42 (CSD refcode KEMJEU) and that of compound 7dfm

were built starting from 7aaa by manual editing using UCSF Chimera package.43 The structures

of enantiomers were generated using an in-house script. Molecular docking was performed

using the GOLD suite40 (CCDC) and the GoldScore scoring function, with the structures 4HL2,44

2OV545 and 4S2P46 as receptors for NDM-1, KPC-2 and OXA-48, respectively. The binding sites

were defined as 15 Å radius spheres centered on the Zn1 ion for NDM-1 and on the OG atom

of Ser70 for KPC-2 and OXA-48. In agreement with our previous studies47–53 showing that an

enhanced conformational search is beneficial, especially for large molecules, a search

efficiency of 200% was used to better explore the ligand conformational space. All other

parameters were used with the default values. Images were generated using UCSF Chimera.43

24

Supporting Information

The Supporting Information contains detailed synthetic procedures and characterizations of

the compounds and copies of the NMR spectra.

Author Information

Corresponding authors:

*E-mail: [email protected]

*E-mail:[email protected].

*E-mail:[email protected].

ORCID:

Alain Pruvost: 0000-0002-7781-7735

Thierry Naas: 0000-0001-9937-9572

Bogdan I. Iorga: 0000-0003-0392-1350

Kevin Cariou: 0000-0002-5854-9632

Author Contributions

This study was conceived by K. Cariou, B. I. Iorga, T. Naas and R. H. Dodd. Synthesis and

characterization of the compounds were performed by E. Romero, M. Benchekroun, S. Ventre,

A. C. A. D’Hollander, K. Vijayakumar and C. Minard. Purification of the proteins, inhibition

assays and MIC assays were performed by S. Oueslati with C. Exilie, L. Tlili and A. Zavala.

Metabolic studies were performed by L. A. Nguyen under the supervision of A. Pruvost.

Structural analysis and molecular modeling studies were performed by A. Zavala, E. Elisée, E.

25

Selwa, P. Retailleau and B. I. Iorga. K. Cariou and B. I. Iorga drafted the manuscript which was

amended and commented on by all authors.

# E. R. & S. O contributed equally; ◊ M. B. & A. C. A. D. contributed equally

.

Conflict of Interest Disclosure: The authors declare no competing financial interest

List of Abbreviations

CTX M 15: Cefotaximase-Munich 15

CMY-2: Cephamycinase-2

DMSO: dimethyl sulfoxideGNB: Gram-negative Bacilli

HCT 116: Human Colorectal Carcinoma cells

KPC: Klebsiella Pneumoniae Carbapenemase

MBL: Metallo-β-lactamase

MIC: Minimum Inhibitory Concentrations

MRC-5: Medical Research Council cell strain 5

ND: Not Determined

NDM: New Delhi metallo-β-lactamase

NE: no effect

OXA: Oxacillinase

SAR: Structure Activity Relationship

SBL: Serine-β-lactamases

26

VIM: Verona Integron metallo-β-lactamase

Acknowledgments

This work was supported by the Laboratory of Excellence in Research on Medication and

Innovative Therapeutics (LERMIT, grant ANR-10-LABX-33 under the program Investissements

d’Avenir ANR-11-IDEX-0003-01), the FCS Campus Paris-Saclay pre-maturation program

(project Inhibase), the SATT Paris-Saclay (project CARBAMAT), the JPIAMR transnational

project DesInMBL (grant ANR-14-JAMR-0002) and the Région Ile-de-France (DIM Malinf). The

authors also thank CNRS, AP-HP, Université Paris-Saclay, and ICSN, for financial support.

References

1. O’Neill, J. (2016). Tackling drug-resistant infections globally: final report and

recommendations. https://amr-

review.org/sites/default/files/160525_Final%20paper_with%20cover.pdf.

2. Nordmann, P., Naas, T., Fortineau, N. and Poirel, L. (2007). Superbugs in the coming new

decade; multidrug resistance and prospects for treatment of Staphylococcus aureus, Enterococcus

spp. and Pseudomonas aeruginosa in 2010. Curr. Opin. Microbiol. 10, 436–440.

3. Nordmann, P., Naas, T. and Poirel, L. (2011). Global spread of carbapenemase-producing

enterobacteriaceae. Emerg. Infect. Dis. 17, 1791–1798.

4. Worthington, R. J. and Melander, C. (2013). Overcoming resistance to β-lactam antibiotics. J.

Org. Chem. 78, 4207–4213.

5. Chellat, M. F., Raguž, L. and Riedl, R. (2016). Targeting Antibiotic Resistance. Angew. Chem. Int.

Ed. 55, 6600–6626.

6. González-Bello, C., Rodríguez, D., Pernas, M., Rodríguez, Á. and Colchón, E. (2019). β-

Lactamase Inhibitors To Restore the Efficacy of Antibiotics against Superbugs. J. Med. Chem. ASAP,

10.1021/acs.jmedchem.9b01279.

27

7. Tooke, C. L., Hinchliffe, P., Bragginton, E. C., Colenso, C. K., Hirvonen, V. H. A., Takebayashi, Y.

and Spencer, J. (2019). β-Lactamases and β-Lactamase Inhibitors in the 21st Century. J. Mol. Biol. 431,

3472–3500.

8. Ehmann, D. E., Jahić, H., Ross, P. L., Gu, R.-F., Hu, J., Kern, G., Walkup, G. K. and Fisher, S. L.

(2012). Avibactam is a covalent, reversible, non–β-lactam β-lactamase inhibitor. Proc. Natl. Acad. Sci.

109, 11663–11668.

9. Wang, D. Y., Abboud, M. I., Markoulides, M. S., Brem, J. and Schofield, C. J. (2016). The road to

avibactam: the first clinically useful non-β-lactam working somewhat like a β-lactam. Future Med.

Chem. 8, 1063–1084.

10. Hecker, S. J., Reddy, K. R., Totrov, M., Hirst, G. C., Lomovskaya, O., Griffith, D. C., King, P.,

Tsivkovski, R., Sun, D., Sabet, M., Tarazi, Z., Clifton, M. C., Atkins, K., Raymond, A., Potts, K. T.,

Abendroth, J., Boyer, S. H., Loutit, J. S., Morgan, E. E., Durso, S. and Dudley, M. N. (2015). Discovery of

a cyclic boronic acid β-lactamase inhibitor (RPX7009) with utility vs class A Serine carbapenemases. J.

Med. Chem. 58, 3682–3692.

11. Hirsch, E. B., Ledesma, K. R., Chang, K.-T., Schwartz, M. S., Motyl, M. R. and Tam, V. H. (2012).

In vitro activity of MK-7655, a novel β-lactamase inhibitor, in combination with imipenem against

carbapenem-resistant Gram-egativne bacteria. Antimicrob. Agents Chemother. 56, 3753–3757.

12. Morinaka, A., Tsutsumi, Y., Yamada, M., Suzuki, K., Watanabe, T., Abe, T., Furuuchi, T., Inamura,

S., Sakamaki, Y., Mitsuhashi, N., Ida, T. and Livermore, D. M. (2015). OP0595, a new diazabicyclooctane:

mode of action as a serine β-lactamase inhibitor, antibiotic and β-lactam ‘enhancer’. J. Antimicrob.

Chemother. 70, 2779–2786.

13. Livermore, D. M., Mushtaq, S., Warner, M., Vickers, A. and Woodford, N. (2017). In vitro activity

of cefepime/zidebactam (WCK 5222) against Gram-negative bacteria. J. Antimicrob. Chemother. 72,

1373–1385.

14. Durand-Réville, T. F., Guler, S., Comita-Prevoir, J., Chen, B., Bifulco, N., Huynh, H., Lahiri, S.,

Shapiro, A. B., McLeod, S. M., Carter, N. M., Moussa, S. H., Velez-Vega, C., Olivier, N. B., McLaughlin,

R., Gao, N., Thresher, J., Palmer, T., Andrews, B., Giacobbe, R. A., Newman, J. V., Ehmann, D. E., de

Jonge, B., O’Donnell, J., Mueller, J. P., Tommasi, R. A. and Miller, A. A. (2017). ETX2514 is a broad-

spectrum β-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including

Acinetobacter baumannii. Nat. Microbiol. 2, 17104.

28

15. Peilleron, L. and Cariou, K. (2020). Synthetic approaches towards avibactam and other

diazabicyclooctane β-lactamase inhibitors. Org. Biomol. Chem. 18, 830–844.

16. Linciano, P., Cendron, L., Gianquinto, E., Spyrakis, F. and Tondi, D. (2019). Ten years with New

Delhi metallo-β-lactamase-1 (NDM-1): from structural insights to inhibitor design. ACS Infect. Dis. 5, 9–

34.

17. Tehrani, K. H. M. E. and Martin, N. I. (2017). Thiol-containing metallo-β-lactamase inhibitors

resensitize resistant Gram-negative bacteria to meropenem. ACS Infect. Dis. 3, 711–717.

18. Klingler, F.-M., Wichelhaus, T. A., Frank, D., Cuesta-Bernal, J., El-Delik, J., Müller, H. F., Sjuts, H.,

Göttig, S., Koenigs, A., Pos, K. M., Pogoryelov, D. and Proschak, E. (2015). Approved drugs containing

thiols as inhibitors of metallo-β-lactamases: strategy to combat multidrug-resistant bacteria. J. Med.

Chem. 58, 3626–3630.

19. King, A. M., Reid-Yu, S. A., Wang, W., King, D. T., De Pascale, G., Strynadka, N. C., Walsh, T. R.,

Coombes, B. K. and Wright, G. D. (2014). Aspergillomarasmine A overcomes metallo-β-lactamase

antibiotic resistance. Nature 510, 503–506.

20. Xiang, Y., Chen, C., Wang, W.-M., Xu, L.-W., Yang, K.-W., Oelschlaeger, P. and He, Y. (2018).

Rhodanine as a potent scaffold for the development of broad-spectrum metallo-β-lactamase

inhibitors. ACS Med. Chem. Lett. 9, 359–364.

21. Brem, J., van Berkel, S. S., Aik, W., Rydzik, A. M., Avison, M. B., Pettinati, I., Umland, K.-D.,

Kawamura, A., Spencer, J., Claridge, T. D. W., McDonough, M. A. and Schofield, C. J. (2014). Rhodanine

hydrolysis leads to potent thioenolate mediated metallo-β-lactamase inhibition. Nat. Chem. 6, 1084–

1090.

22. Leiris, S., Coelho, A., Castandet, J., Bayet, M., Lozano, C., Bougnon, J., Bousquet, J., Everett, M.,

Lemonnier, M., Sprynski, N., Zalacain, M., Pallin, T. D., Cramp, M. C., Jennings, N., Raphy, G., Jones, M.

W., Pattipati, R., Shankar, B., Sivasubrahmanyam, R., Soodhagani, A. K., Juventhala, R. R., Pottabathini,

N., Pothukanuri, S., Benvenuti, M., Pozzi, C., Mangani, S., De Luca, F., Cerboni, G., Docquier, J.-D. and

Davies, D. T. (2019). SAR studies leading to the identification of a novel series of metallo-β-lactamase

inhibitors for the treatment of carbapenem-resistant enterobacteriaceae infections that display

efficacy in an animal infection model. ACS Infect. Dis. 5, 131–140.

23. Yu, Z.-J., Liu, S., Zhou, S., Li, H., Yang, F., Yang, L.-L., Wu, Y., Guo, L. and Li, G.-B. (2018). Virtual

29

target screening reveals rosmarinic acid and salvianolic acid A inhibiting metallo- and serine-β-

lactamases. Bioorg. Med. Chem. Lett. 28, 1037–1042.

24. Brem, J., Cain, R., Cahill, S., McDonough, M. A., Clifton, I. J., Jiménez-Castellanos, J.-C., Avison,

M. B., Spencer, J., Fishwick, C. W. G. and Schofield, C. J. (2016). Structural basis of metallo-β-lactamase,

serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates. Nat. Commun. 7, 1–8.

25. Cahill, S. T., Cain, R., Wang, D. Y., Lohans, C. T., Wareham, D. W., Oswin, H. P., Mohammed, J.,

Spencer, J., Fishwick, C. W. G., McDonough, M. A., Schofield, C. J. and Brem, J. (2017). Cyclic boronates

inhibit all classes of β-lactamases. Antimicrob. Agents Chemother. 61, e02260-16.

26. Krajnc, A., Brem, J., Hinchliffe, P., Calvopiña, K., Panduwawala, T. D., Lang, P. A., Kamps, J. J. A.

G., Tyrrell, J. M., Widlake, E., Saward, B. G., Walsh, T. R., Spencer, J. and Schofield, C. J. (2019). Bicyclic

boronate VNRX-5133 inhibits metallo- and serine-β-lactamases. J. Med. Chem. 62, 8544–8556.

27. Parkova, A., Lucic, A., Krajnc, A., Brem, J., Calvopina, K., Langley, G., McDonough, M. A.,

Trapencieris, P. and Schofield, C. J. (2019). Broad spectrum β-lactamase inhibition by a thioether

substituted bicyclic boronate. ACS Infect. Dis. ASAP, 10.1021/acsinfecdis.9b00330.

28. Wang, Y.-L., Liu, S., Yu, Z.-J., Lei, Y., Huang, M.-Y., Yan, Y.-H., Ma, Q., Zheng, Y., Deng, H., Sun,

Y., Wu, C., Yu, Y., Chen, Q., Wang, Z., Wu, Y. and Li, G.-B. (2019). Structure-based development of (1-

(3′-mercaptopropanamido)methyl)boronic acid derived broad-spectrum, dual-action inhibitors of

metallo- and serine-β-lactamases. J. Med. Chem. 62, 7160–7184.

29. Pitts, C. R. and Lectka, T. (2014). Chemical synthesis of β-lactams: asymmetric catalysis and

other recent advances. Chem. Rev. 114, 7930–7953.

30. Staudinger, H. (1907). Zur Kenntniss der Ketene. Diphenylketen. Justus Liebigs Ann. Chem. 356,

51–123.

31. DeKorver, K. A., Li, H., Lohse, A. G., Hayashi, R., Lu, Z., Zhang, Y. and Hsung, R. P. (2010).

Ynamides: A modern functional group for the new millennium. Chem. Rev. 110, 5064–5106.

32. Evano, G., Coste, A. and Jouvin, K. (2010). Ynamides: Versatile tools in organic synthesis.

Angew. Chem. Int. Ed. 49, 2840–2859.

33. Lu, P. and Wang, Y. (2012). The thriving chemistry of ketenimines. Chem. Soc. Rev. 41, 5687–

5705.

30

34. Dodd, R. H. and Cariou, K. (2018). Ketenimines Generated from Ynamides: Versatile Building

Blocks for Nitrogen-Containing Scaffolds. Chem. – Eur. J. 24, 2297–2304.

35. Hentz, A., Retailleau, P., Gandon, V., Cariou, K. and Dodd, R. H. (2014). Transition-metal-free

tunable chemoselective N-functionalization of indoles with ynamides. Angew. Chem. Int. Ed. 126,

8473–8477.

36. Romero, E., Minard, C., Benchekroun, M., Ventre, S., Retailleau, P., Dodd, R. H. and Cariou, K.

(2017). Base-Mediated Generation of Ketenimines from Ynamides: Direct Access to Azetidinimines by

an Imino-Staudinger Synthesis. Chem. – Eur. J. 23, 12991–12994.

37. Van Camp, A., Goossens, D., Moya-Portuguez, M., Marchand-Brynaert, J. and Ghosez, L. (1980).

Synthesis of N-(tosyl)azetidin-2-imines. Tetrahedron Lett. 21, 3081–3084.

38. Whiting, M. and Fokin, V. V. (2006). Copper-catalyzed reaction cascade: direct conversion of

alkynes into N-sulfonylazetidin-2-imines. Angew. Chem. Int. Ed. 45, 3157–3161.

39. Bonnin, R. A., Poirel, L., Carattoli, A. and Nordmann, P. (2012). Characterization of an IncFII

Plasmid Encoding NDM-1 from Escherichia coli ST131. PLOS ONE 7, e34752.

40. Verdonk, M. L., Cole, J. C., Hartshorn, M. J., Murray, C. W. and Taylor, R. D. (2003). Improved

protein–ligand docking using GOLD. Proteins Struct. Funct. Bioinforma. 52, 609–623.

41. Artimo, P., Jonnalagedda, M., Arnold, K., Baratin, D., Csardi, G., de Castro, E., Duvaud, S., Flegel,

V., Fortier, A., Gasteiger, E., Grosdidier, A., Hernandez, C., Ioannidis, V., Kuznetsov, D., Liechti, R.,

Moretti, S., Mostaguir, K., Redaschi, N., Rossier, G., Xenarios, I. and Stockinger, H. (2012). ExPASy: SIB

bioinformatics resource portal. Nucleic Acids Res. 40, W597–W603.

42. Groom, C. R. and Allen, F. H. (2014). The Cambridge Structural Database in Retrospect and

Prospect. Angew. Chem. Int. Ed. 53, 662–671.

43. Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. and

Ferrin, T. E. (2004). UCSF Chimera—A visualization system for exploratory research and analysis. J.

Comput. Chem. 25, 1605–1612.

44. Kim, Y., Cunningham, M. A., Mire, J., Tesar, C., Sacchettini, J. and Joachimiak, A. (2013). NDM-

1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism. FASEB J. 27,

1917–1927.

31

45. Ke, W., Bethel, C. R., Thomson, J. M., Bonomo, R. A. and van den Akker, F. (2007). Crystal

Structure of KPC-2: Insights into Carbapenemase Activity in Class A β-Lactamases. Biochemistry 46,

5732–5740.

46. King, D. T., King, A. M., Lal, S. M., Wright, G. D. and Strynadka, N. C. J. (2015). Molecular

Mechanism of Avibactam-Mediated β-Lactamase Inhibition. ACS Infect. Dis. 1, 175–184.

47. Surpateanu, G. and Iorga, B. I. (2012). Evaluation of docking performance in a blinded virtual

screening of fragment-like trypsin inhibitors. J. Comput. Aided Mol. Des. 26, 595–601.

48. Colas, C. and Iorga, B. I. (2014). Virtual screening of the SAMPL4 blinded HIV integrase

inhibitors dataset. J. Comput. Aided Mol. Des. 28, 455–462.

49. Martiny, V. Y., Martz, F., Selwa, E. and Iorga, B. I. (2016). Blind Pose Prediction, Scoring, and

Affinity Ranking of the CSAR 2014 Dataset. J. Chem. Inf. Model. 56, 996–1003.

50. Selwa, E., Martiny, V. Y. and Iorga, B. I. (2016). Molecular docking performance evaluated on

the D3R Grand Challenge 2015 drug-like ligand datasets. J. Comput. Aided Mol. Des. 30, 829–839.

51. Selwa, E., Elisée, E., Zavala, A. and Iorga, B. I. (2018). Blinded evaluation of farnesoid X receptor

(FXR) ligands binding using molecular docking and free energy calculations. J. Comput. Aided Mol. Des.

32, 273–286.

52. Chaput, L., Selwa, E., Elisée, E. and Iorga, B. I. (2019). Blinded evaluation of cathepsin S

inhibitors from the D3RGC3 dataset using molecular docking and free energy calculations. J. Comput.

Aided Mol. Des. 33, 93–103.

53. Elisée, E., Gapsys, V., Mele, N., Chaput, L., Selwa, E., de Groot, B. L. and Iorga, B. I. (2019).

Performance evaluation of molecular docking and free energy calculations protocols using the D3R

Grand Challenge 4 dataset. J. Comput. Aided Mol. Des. 33, 1031–1043.

download fileview on ChemRxivmanuscriptAZETI.pdf (1.86 MiB)



S1

SUPPLEMENTARY INFORMATIONS FOR:

Azetidinimines as a novel series of non-covalent broad-spectrum

inhibitors of β-lactamases with submicromolar activities against

carbapenemases of classes A, B and D.

Eugénie Romero,†,# Saoussen Oueslati,‡,‖,# Mohamed Benchekroun,†,◊ Agathe C. A. D’Hollander,†,◊

Sandrine Ventre,† Kamsana Vijayakumar,† Corinne Minard,† Cynthia Exilie,‡,‖ Linda Tlili,‡,‖ Pascal

Retailleau,† Agustin Zavala,† Eddy Elisée,† Edithe Selwa,† Laetitia A. Nguyen,§ Alain Pruvost,§ Thierry

Naas, *,‡,‖,Ʇ,¶,◊ Bogdan I. Iorga,*,† Robert H. Dodd,† Kevin Cariou*,†,○

† Université Paris-Saclay, CNRS, Institut de Chimie des Substances Naturelles, LabEx LERMIT, UPR 2301, 91198, Gif-sur-Yvette,

France.

‡ UMR1184, Inserm, Université Paris-Saclay, LabEx LERMIT, Hôpital Bicêtre, Le Kremlin-Bicêtre, France.

‖ Bacteriology-Hygiene Unit, Hôpital Bicêtre, Le Kremlin-Bicêtre, France.

§ Université Paris-Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé, Gif-sur-Yvette, France.

Ʇ EERA Unit "Evolution and Ecology of Resistance to Antibiotics Unit, Institut Pasteur-AP-HP-Université Paris-Saclay, Paris,

France.

¶ Associated French National Reference Center for Antibiotic Resistance: Carbapenemase-Producing Enterobacteriaceae, Le

Kremlin-Bicêtre, France.

○ Current address: Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for

Inorganic Chemical Biology, 75005 Paris, France.

S2

Supplementary information contains:

1. General Remarks ....................................................................................................... S3

2. General Procedures ................................................................................................... S3

3. Preparation and Analytical Data of Azetidinimines in Table 1 .................................... S5

4. Preparation and Analytical Data of Azetidinimines in Table 2 .................................. S14




8. NMR Spectra of Azetidinimines in Table 1 ............................................................... S39

9. NMR Spectra of Azetidinimines in Table 2 ............................................................... S53

10. NMR Spectra of Azetidinimines in Table 3 ............................................................ S60



S3

1. General Remarks

Melting points were measured in capillary tubes on a Büchi B-540 apparatus and were uncorrected.

Infrared spectra were recorded on a Perkin Elmer Spectrum BX FT-IR spectrometer. Proton (1H) and

carbon (13C) NMR spectra were recorded on Bruker spectrometers: Avance 300 MHz (QNP - 13C, 31P,

19F - probe or Dual 13C probe) and Avance 500 MHz (BB0 - ATM probe or BBI - ATM probe). Carbon

NMR (13C) spectra were recorded at 125 or 75 MHz, using a broadband decoupled mode with the

multiplicities obtained using a DEPT sequence. NMR experiments were carried out in

deuterochloroform (CDCl3), in deuterobenzene (C6D6) and in deuteromethanol (CD3OD); chemical

shifts (δ) are reported in parts per million δ with reference to the residual solvent peak: CDCl3 (1H: 7.26;

13C: 77.16), C6D6 (1H: 7.15; 13C: 128.6) or CD3OD (1H: 3.31; 13C: 49.0). The following abbreviations were

used for the proton spectra multiplicities: s: singlet, bs: broad singlet, d: doublet, t: triplet, q: quartet,

m: multiplet, br: broad. Coupling constants (J) are reported in Hertz (Hz). Mass spectra were obtained

either with a LCT (Micromass) instrument using electrospray ionization (ES), or from a Time of Flight

analyzer (ESI-MS) for the high resolution mass spectra (HRMS). Thin-layer chromatography was

performed on silica gel 60 F254 on aluminum plates (Merck) and visualized under a UVP Mineralight

UVLS-28 lamp (254 nm) and with 4-anisaldehyde and phosphomolybdic acid stains in ethanol. Flash

chromatography was conducted on Merck silica gel 60 (40-63 μm) at medium pressure (300 mbar).

Unless stated otherwise, all reagents were obtained from commercial suppliers. When necessary,

organic solvents were dried and/or distilled prior to use and stored over molecular sieves under

nitrogen. Extra dry DMF and t-BuOLi (2.2 M solution in THF) were purchased from Acros Organic as

AcroSeal® reagents. Microwave reactions were performed using either a Monowave 300® or a

Monowave 50® instrument manufactured and distributed by Anton-Paar.

2. General Procedures

General Procedure for the Synthesis of the Starting Ynamides 31

Step 1: To a solution of tert-butyl arylcarbamate (1.0 equiv.) in toluene (0.5 M) were added, under an

argon atmosphere, tribasic potassium phosphate (2.4 equiv.), 1,10-phenanthroline (40 mol%) and

1 (a) Zhang, Y.; Hsung, R. P.; Tracey, M. R.; Kurtz, K. C. M.; Vera, E. L. Org. Lett. 2004, 6, 1151. (b) Dooleweerdt, K.; Birkedal, H.; Ruhland, T.; Skrydstrup T. J. Org. Chem. 73, 2008, 9447.

S4

pentahydrated copper sulfate (20 mol%). (Bromoethynyl)triisopropylsilane (1.0 equiv.) in toluene (0.6

M) was then added and the resulting mixture was stirred at 100 °C for 4 days. After completion of the

reaction, the mixture was cooled to room temperature, EtOAc was added and the mixture was filtered

over a pad of silica and concentrated in vacuo. The crude residue was purified by flash chromatography

on SiO2 (first petroleum ether then 5% EtOAc in petroleum ether) to provide tert-butyl

aryl((triisopropylsilyl)ethynyl)carbamate.

Step 2: To a solution of tert-butyl aryl((triisopropylsilyl)ethynyl)carbamate (1.0 equiv.) in THF held at -

0 °C was introduced TBAF (1 M in THF, 2.0 equiv.) and the reaction mixture was stirred for 5 min at -

10 °C. The reaction was then quenched with saturated ammonium chloride solution and extracted with

MTBE. The organic extract was washed with brine, dried (MgSO4), filtered and concentrated under

reduced pressure. The ynamide was purified by flash chromatography on SiO2 (1% EtOAc in petroleum

ether) to provide tert-butyl ethynyl(phenyl)carbamate.

Spectral data for ynamides 3a–c2 3d–f3 were in agreement with the previously reported literature data.

General Procedure for the Synthesis of the Starting Imines 6

To a solution of benzylamine (1.0 equiv) in EtOH (7.8 mL per mmol) was added the corresponding

aldehyde (1.0 equiv). The reaction was sonicated for 60 min before being concentrated in vacuo. Water

was added and the organics were extracted with DCM, combined, dried over MgSO4, filtered and

concentrated in vacuo to afford the desired pure imine.

General Procedure for Azetidinimines Synthesis

The imine (1.0 equiv.), the ynamide (2.0 equiv.) and the additive – silica gel (1.0 equiv.) or ZnOTf2 (10

mol%) – were successively added in a microwave sealable tube and placed under argon before the

addition of t-BuOLi – solid or 2.2 M in solution in THF – (2.0 equiv.) followed by extra dry DMF (0.3M).4

The sealed tube was placed in a microwave apparatus for 1 h at 100 °C. The crude material was purified

by flash chromatography on silica gel or with preparative TLC.

2 (a) for 1a: Hentz, A.; Retailleau, P.; Gandon, V.; Cariou, K.; Dodd, R. H. Angew. Chem., Int. Ed. 2014, 53, 8333. (b) for 1b,c: Huang, H.; Tang, L.; Han, X.; He, G.; Xi, Y.; Zhu, H. Chem. Commun. 2016, 52, 4321. 3 Romero, E., Minard, C., Benchekroun, M., Ventre, S., Retailleau, P., Dodd, R. H. and Cariou, K. Chem. – Eur. J. 2017, 23, 12991. 4 At this stage the mixture generally develops a deep dark brown/red color. If not, this is usually a sign that either the solvent or the base is not of optimal quality and that the reaction might fail.

S5

General procedure for demethylation with BBr3

An argon-flushed and stirred solution of azetidinimine in anhydrous dichloromethane ([C]~0.1 M) is

cooled at -78°C then BBr3 (1M solution in DCM, 4 equiv.) is added dropwise. The solution is further

stirred at -78°C for 2h then allowed to warm to rt. Once the reaction completed (TLC monitoring), the

reaction mixture is carefully quenched at -78°C with a 1:1 mixture of MeOH/DCM (10 mL). After

warming to rt, the solvents are evaporated then the crude residue is resolubilized in DCM, washed

with a saturated aqueous solution of NaHCO3 and water. The organic layer is dried on sodium sulfate,

filtered then evaporated under reduced pressure. The resulting residue is subsequently purified by

automated flash chromatography using a gradient of AcOEt in heptane (AcOEt 0%60% over 30 min).

3. Preparation and Analytical Data of Azetidinimines in Table 1

1-(4-methoxyphenyl)-N-4-diphenylazetidin-2-imine 7aaa

(MW = 328.415 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6aa

(63 mg, 0.3 mmol, 1.0 equiv.), ynamide 3a (130 mg, 0.6 mmol, 2.0 equiv.), t-BuOLi (48 mg, 0.6 mmol,

2.0 equiv.) and SiO2 (18 mg, 0.3 mmol, 1.0 equiv.) in DMF (900 µL). The crude material was purified by

flash chromatography on silica gel (10% ethyl acetate in heptane as eluent) to provide the

corresponding azetidinimine with 54 % yield (53 mg, yellow solid).

1H NMR (300 MHz, CDCl3): δ 7.37-7.16 (m, 9H), 6.99-6.92 (m, 3H), 6.72 (d, J = 9.1 Hz, 2H), 5.07 (dd, J =

6.1, 2.9 Hz, 1H), 3.66 (s, 3H), 3.42 (dd, J = 14.6, 6.1 Hz, 1H), 2.86 (dd, J = 14.6, 2.9 Hz, 1H).

13C NMR (75 MHz, CDCl3): δ 154.8 (C), 154.1 (C), 148.6 (C), 139.4 (C), 133.7 (C), 129.1 (2CH), 128.9

(2CH), 128.3 (CH), 125.9 (2CH), 122.8 (CH), 122.2 (2CH), 117.5 (2CH), 114.2 (2CH), 58.2 (CH), 55.5 (CH3),

40.5 (CH2).

HRMS-ESI (m/z) calcd for C22H21N2O [M+H]+ 329.1648, found 329.1658.

IR (neat): 2993, 2931, 2894, 1668, 1592, 1510, 1486, 1390, 1256, 1241, 1162, 1035 cm-1.

S6

m.p. (pentane/Et2O): 112.0 °C

1-(4-methoxyphenyl)-N-4-diphenylazetidin-2-imine 7aab

(MW = 342.442 g.mol-1)





corresponding azetidinimine with 70 % yield (52 mg, beige solid).

1H NMR (300 MHz, CDCl3): δ .47-7.28 (m, 9H), 7.09-6.99 (m, 3H), 6.81 (d, J = 8.8 Hz, 2H), 5.20-5.13 (m,

1H), 3.98 (q, J = 7.1 Hz, 2H), 3.51 (dd, J = 14.3, 6.4 Hz, 1H), 2.95 (dd, J = 14.8, 2.9 Hz, 1H), 1.38 (t, J = 7.1

Hz, 3H).

13C NMR (75 MHz, CDCl3): δ 154.1 (C), 150.9 (C), 148.4 (C), 133.3 (C), 129.1 (2CH), 128.9 (2CH), 128.3

(CH), 127.6 (C), 125.9 (2CH), 122.8 (CH), 122.2 (2CH), 117.5 (2CH), 114.9 (2CH), 63.7 (CH2), 58.1 (CH),

40.7 (CH2), 14.9 (CH3).


IR (neat): 2982, 2875, 1672, 1591, 1510, 1475, 1390, 1241, 1162, 1045 cm-1.

m.p: 126.0 °C

1-(4-benzyloxyphenyl)-N-4-diphenylazetidin-2-imine 7aac

(MW = 404.5130 g.mol-1)

S7

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ac

(58 mg, 0.2 mmol, 1 equiv.), ynamide 3a (86 mg, 0.4 mmol, 2 equiv.), t-BuOLi (180 µL, 0.4 mmol, 2

equiv.) and zinc trifluoromethanesulfonate (7.3 mg, 0.02 mmol, 10 mol%) in DMF (420 µL). The crude

material was purified by flash chromatography on silica gel (10% ethyl acetate in heptane as eluent) to

provide the corresponding azetidinimine with 70 % yield (52 mg, brown foam).

1H NMR (300 MHz, CDCl3): δ 7.49 – 7.20 (m, 14H), 7.11 – 6.98 (m, 3H), 6.93 – 6.81 (m, 2H), 5.15 (dd, J

= 6.0, 2.9 Hz, 1H), 5.00 (s, 2H), 3.5 (dd, J = 14.6, 6.0 Hz, 1H), 2.94 (dd, J = 14.6, 2.9 Hz, 1H).

13C NMR (75 MHz, CDCl3): δ 154.2 (2C), 148.7 (C), 139.5 (C), 137.3 (C), 134.0 (C), 129.2 (2CH), 129.1

(2CH), 128.7 (2CH), 128.5 (CH), 128.0 (CH), 127.6 (2CH), 126.1 (2CH), 123.0 (CH), 122.4 (2CH), 117.6

(2CH), 115.5 (2CH), 70.5 (CH2), 58.3 (CH), 40.6 (CH2).

HRMS-ESI (m/z) calcd for C21H18IN2 [(M+H)+] 425.0515, found 425.0577.

IR (neat): 3064, 3037, 2923, 1678, 1583, 1483, 1455, 1411, 1380, 1242, 1161, 1065 cm-1.

m.p: 151.0 °C

1-(3-methoxyphenyl)-N-4-diphenylazetidin-2-imine 7aad

(MW = 328.415 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ad

(42 mg, 0.2 mmol, 1.0 equiv.), ynamide 3a (87 mg, 0.4 mmol, 2.0 equiv.), t-BuOLi (180 µL, 0.4 mmol,

2.0 equiv.) and zinc trifluoromethanesulfonate (7.3 mg, 0.02 mmol, 10 mol%) in DMF (420 µL). The

crude material was purified by flash chromatography on silica gel (10% ethyl acetate in heptane as

eluent) to provide the corresponding azetidinimine with 29 % yield (19 mg, yellow wax).

1H NMR (300 MHz, CDCl3): δ 7.45 – 7.21 (m, 8H), 7.14 (t, J = 8.1 Hz, 1H), 7.09 – 7.00 (m, 3H), 6.96 –

6.89 (m, 1H), 6.53 (ddd, J = 8.1, 2.5, 0.8 Hz, 1H), 5.17 (dd, J = 6.2, 3.0 Hz, 1H), 3.75 (s, 3H), 3.51 (dd, J =

14.8, 6.2 Hz, 1H), 2.95 (dd, J = 14.8, 3.0 Hz, 1H).

S8


(2CH), 129.1 (CH), 128.5 (CH), 126.0 (2CH), 123.2 (CH), 122.3 (2CH), 108.8 (CH), 108.1 (CH), 102.6 (CH),

58.4 (CH), 55.3(CH3), 40.6 (CH2).


IR (neat): 3060, 3030, 2922, 2835, 1671, 1587, 1486, 1467, 1455, 1376, 1327, 1305, 1271, 1243, 1224,

1206, 1192, 1178, 1152, 1089, 1071, 1043 cm-1.

1-(3,4-dimethoxyphenyl)-N-4-diphenylazetidin-2-imine 7aaf

(MW = 358.4410 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6af




eluent) to provide the corresponding azetidinimine with 36 % yield (26 mg, yellow foam).

1H NMR (300 MHz, CDCl3): δ 7.52 (d, J = 2.3 Hz, 1H), 7.48 – 7.22 (m, 7H), 7.06 – 6.99 (m, 3H), 6.72 (d, J

= 8.6 Hz, 1H), 6.64 (dd, J = 8.6, 2.3 Hz, 1H), 5.15 (dd, J = 6.1, 2.9 Hz, 1H), 3.8 (2 overlaps s, 6H), 3.52 (dd,

J = 14.6, 6.1 Hz, 1H), 2.97 (dd, J = 14.7, 2.9 Hz, 1H).

13C NMR (75 MHz, CDCl3): δ 154.2 (C), 149.4 (C), 148.7 (C), 144.4 (C), 139.5 (C), 134.3 (C), 129.2 (2CH),

129.1 (2CH), 128.5 (CH), 126.1 (2CH), 123.0 (2CH), 122.3 (CH), 111.7 (CH), 107.3 (CH), 102.0 (CH), 58.5

(CH), 56.3 (CH3), 55.9 (CH3), 40.7 (CH2).

HRMS-ESI (m/z) calcd for C23H23N2O2 [(M+H)+] 359.1760, found 359.1763.

IR (neat): 2999, 2829, 2832, 1665, 1610, 1589, 1510, 1487, 1454, 1397, 1357, 1322, 1264, 1235, 1192,

1176, 1153, 1069, 1024, 984 cm-1.

S9

1-(benzo[d][1,3]dioxol-5-yl)-N,4-diphenylazetidin-2-imine 7aag

(MW = 342.3980 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6af





1H NMR (300 MHz, CDCl3): 7.46 – 7.22 (m, 8H), 7.12 – 6.97 (m, 3H), 6.77 (dd, J = 8.4, 2.1 Hz, 1H), 6.67

(d, J = 8.4 Hz, 1H), 5.88 (dd, J = 2.7, 1.4 Hz, 2H), 5.12 (dd, J = 6.1, 2.9 Hz, 1H), 3.50 (dd, J = 14.7, 6.1 Hz,

1H), 2.93 (dd, J = 14.7, 2.9 Hz, 1H).


129.1 (2CH), 128.5 (CH), 126.0 (2CH), 123.1 (CH), 122.3 (2CH), 108.8 (CH), 108.3 (CH), 101.1(CH), 99.3

(CH2), 58.6 (CH), 40.6 (CH2).


IR (neat): 3030, 2920, 1667, 1631, 1590, 1500, 1481, 1454, 1442, 1380, 1339, 1308, 1287, 1241, 1218,

1205, 1164, 1139, 1113, 1096, 1062, 1036 cm-1.

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-4-diphenylazetidin-2-imine 7aah

(MW = 356.4250 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ag


S10




1H NMR (300 MHz, CDCl3): 7.49 – 7.22 (m, 7H), 7.08 – 6.98 (m, 4H), 6.95 (dd, J = 8. 7, 2.5, 1H), 6.74 (d,

J = 8.7 Hz, 1H), 5.11 (dd, J = 6.1, 2.9 Hz, 1H), 4.27 – 4.13 (m, 4H), 3.48 (dd, J = 14.7, 6.1 Hz, 1H), 2.92 (dd,

J = 14.7, 2.9 Hz, 1H).


129.0 (2CH), 128.5 (CH), 126.0 (2CH), 123.0 (CH), 122.3 (2CH), 117.4 (CH), 110.0 (CH), 105.8 (CH), 64.6

(CH2), 64.4 (CH2), 58.4 (CH), 40.7(CH2).


IR (neat): 3029, 2921, 2870, 1660, 1619, 1588, 1505, 1485, 1455, 1397, 1351, 1308, 1279, 1240, 1223,

1209, 1190, 1155, 1127, 1065, 1027 cm-1.

N,4-diphenyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-imine 7aai

(MW = 388.4670 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ai




corresponding azetidinimine with 54 % yield (22 mg, light brown solid).

1H NMR (300 MHz, CDCl3): δ 7.51-7.26 (m, 7H), 7.12-7.01 (m, 3H), 6.78 (s, 2H), 5.16 (dd, J = 6.0, 2.9 Hz,

1H), 3.79 (s, 3H), 3.73 (s, 6H), 3.54 (dd, J = 14.8, 6.0 Hz, 1H), 3.00 (dd, J = 14.8, 2.9 Hz, 1H).

13C NMR (75 MHz, CDCl3): δ 153.4 (2C), 148.0 (C), 136.2 (C), 132.8 (C), 129.1 (2CH), 129.0 (2CH), 128.5

(2CH), 126.0 (2CH), 125.0 (C), 123.0 (CH), 122.2 (C), 122.1 (CH), 94.2 (2CH), 60.9 (CH), 58.6 (CH3), 56.0

(2CH3), 40.4 (CH2). Quaternary carbons signals were obtained from the HMBC spectrum.

S11

HRMS-ESI (m/z) calcd for C24H25N2O3 [M+H]+ 389.1860, found 389.1871.

IR (neat): 2970, 2926, 1739, 1693, 1599, 1584, 1449, 1229, 1207, 1110, 1015 cm-1.

1-phenyl-N-4-diphenylazetidin-2-imine 7aaj

(MW= 298.38 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6aj

(36.2 mg, 0.2 mmol, 1.0 equiv.), ynamide 3a (86.9 mg, 0.4 mmol, 2.0 equiv.), t-BuOLi (450 µL, 0.4 mmol,



eluent) to provide the corresponding azetidinimine with 22 % yield (13.3 mg, yellow foam).

1H NMR (300 MHz, CDCl3): δ 7.48-7.23 (m, 12H), 7.08-6.95 (m, 3H), 5.19 (dd, J = 6.4, 2.8 Hz, 1H), 3.52

(dd, J = 14.8, 6.4 Hz, 1H), 2.96 (dd, J = 14.8, 2.8 Hz, 1H).

13C NMR (75 MHz, CDCl3): δ 154.5 (C), 148.5 (C), 140.0 (C), 139.4 (C), 129.2 (CH), 129.1 (2CH), 129.0

(2CH), 128.5 (CH), 126.0 (2CH), 123.2 (CH), 122.3 (2CH), 116.6 (2CH), 116.5 (2CH), 58.2 (CH), 40.5 (CH2).

HRMS-ESI (m/z) calcd for C21H19N2 [(M+H)+] 299.1543, found 299.1563.

IR (neat): 3068, 2955, 2918, 2850, 1677, 1587, 1498, 1482, 1383 cm-1.

1-(4-iodophenyl)-N-4-diphenylazetidin-2-imine 7aak

(MW = 424.2855 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ak

(61 mg, 0.2 mmol, 1 equiv.), ynamide 3a (86 mg, 0.4 mmol, 2 equiv.), t-BuOLi (180 µL, 0.4 mmol, 2

S12

equiv.) and zinc trifluoromethanesulfonate (7.3 mg, 0.02 mmol, 10 mol%) in DMF (420 µL). The crude

material was purified by flash chromatography on silica gel (10% ethyl acetate in heptane as eluent) to

provide the corresponding azetidinimine with 36 % yield (17 mg, beige solid).

1H NMR (300 MHz, CDCl3): δ 7.10 (d, J = 8.8 Hz, 2H), 7.32 – 7.06 (m, 9H), 6.98 – 6.84 (m, 3H), 5.03 (dd,

J = 6.2, 3.0 Hz, 1H), 3.39 (dd, J = 14.8, 6.2 Hz, 1H), 2.82 (dd, J = 14.8, 3.0 Hz, 1H).

13C NMR (75 MHz, CDCl3): δ 154.2 (C), 148.2 (C), 139.5 (C), 138.9 (C), 137.8 (2 CH), 129.3 (2 CH), 129.2

(2 CH), 128.7 (CH), 126.0 (2 CH), 123.4 (CH), 122.2 (2 CH), 118.5 (2 CH), 84.9 (C), 58.3 (CH), 40.8(CH2).

HRMS-ESI (m/z) calcd for C21H18IN2 [(M+H)+] 425.0515, found 425.0577.

IR (neat): 3064, 3037, 2923, 1678, 1583, 1483, 1455, 1411, 1380, 1242, 1161, 1065 cm-1.

m.p: 151.0 °C

1-(4-(methylthio)phenyl)-N,4-diphenylazetidin-2-imine 7aal

(MW = 344.1347 g.mol-1)





eluent) to provide the corresponding azetidinimine with 30 % yield (21 mg, yellow solid).

1H NMR (300 MHz, CDCl3): δ 7.36 – 7.14 (m, 9H), 7.14 – 7.06 (m, 2H), 7.00 – 6.89 (m, 3H), 5.07 (dd, J =

6.2, 3.0 Hz, 1H), 3.41 (dd, J = 14.8, 6.2 Hz, 1H), 2.85 (dd, J = 14.8, 3.0 Hz, 1H), 2.32 (s, 3H).


(2CH), 128.8 (2CH), 128.4 (CH), 126.0 (2CH), 123.2 (CH), 122.2 (2CH), 117.1 (2CH), 58.3 (CH), 40.7 (CH2),

17.4 (CH3).

HRMS-ESI (m/z) calcd for C22H21N2S [(M+H)+] 345.1347, found 345.1451.

S13

4-(2-phenyl-4-(phenylimino)azetidin-1-yl)phenol 7aam

(MW = 344.1347 g.mol-1)

Prepared according to the general procedure for demethylation with BBr3 with 7aaa (100 mg, 0.3

mmol, 1.0 equiv.), 1M BBr3 in DCM (1.2 mL, 1.2 mmol, 4.0 equiv.) in anhydrous DCM (2 mL). Yellow oil.

(36 mg, 38%).

1H NMR (300 MHz, CDCl3): δ 7.41 – 7.23 (m, 10 H), 7.07 – 7.03 (m, 3 H), 6.68 (d, J = 8.9 Hz, 2H), 5.78

(bs, 1H), 5.16 (dd, J = 6.0, 2.9 Hz, 1H), 3.50 (dd, J = 14.7, 6.0 Hz, 1H), 2.92 (dd, J = 14.7, 2.9 Hz, 1H).


(CH), 126.1 (2CH), 123.3 (CH), 122.5 (2CH), 118.5 (2CH), 116.0 (2CH), 58.6 (CH), 40.4 (CH2).

HRMS-ESI (m/z) calcd for C21H19N2O [(M+H)+] 315.1497, found 315.1503.

IR (neat): 3030, 2920, 2851, 1662, 1590, 1510, 1487, 1455, 1390, 1356, 1304, 1234, 1192, 1159, 1109,

1065, 1027 cm-1.

S14


4-(4-chlorophenyl)-1-(4-methoxyphenyl)-N-phenylazetidin-2-imine 7aba

(MW = 362.8570 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ba




corresponding azetidinimine with 64 % yield (23 mg, yellow solid).

1H NMR (300 MHz, CDCl3): δ 7.34-7.25 (m, 6H), 7.24-7.16 (m, 2H), 6.99-6.90 (m, 3H), 6.73 (d, J = 9.1

Hz, 2H), 5.04 (dd, J = 6.0, 2.8 Hz, 1H), 3.67 (s, 3H), 3.42 (dd, J = 14.6, 6.0 Hz, 1H), 2.81 (dd, J = 14.6, 2.8

Hz, 1H).


129.0 (2CH), 127.3 (2CH), 123.0 (CH), 122.2 (2CH), 117.5 (2CH), 114.3 (2CH), 57.5 (CH), 55.5 (CH3), 40.5

(CH2).

HRMS-ESI (m/z) calcd for C22H2035ClNO2 [M+H]+ 363.1259, found 363.1246; calcd for C22H20

37ClNO2

[M+H]+ 365.1229, found 365.1251.

IR (neat): 3362, 2909, 2836, 1671, 1590, 1485, 1385, 1295, 1253, 1237, 1162, 1034, 821 cm-1.

m.p. 138.7 °C.

1,4-bis(4-methoxyphenyl)-N-phenylazetidin-2-imine 7aca

S15

(MW = 358.4410 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ca




corresponding azetidinimine with 40 % yield (14 mg, light brown solid).

1H NMR (300 MHz, CDCl3): δ 7.42-7.25 (m, 6H), 7.05-6.96 (m, 3H), 6.88 (d, J = 8.6 Hz, 2H), 6.78 (d, J =

8.9 Hz, 2H), 5.08 (dd, J = 5.6, 3.0 Hz, 1H), 3.78 (s, 3H), 3.72 (s, 3H), 3.45 (dd, J = 14.7, 5.6 Hz, 1H), 2.90

(dd, J = 14.7, 3.0 Hz, 1H).


127.4 (2CH), 122.9 (CH), 122.4 (2CH), 117.7 (2CH), 114.6 (2CH), 114.4 (2CH), 58.1 (CH), 55.6 (CH3), 55.5

(CH3), 40.8 (CH2).

HRMS-ESI (m/z) calcd for C23H23N2O2 [M+H]+ 359.1754, found 359.1752.

IR (neat): 2926, 1720, 1609, 1365, 1217, 1134 cm-1.

m.p. 78.0 °C.

1,4-bis(4-methoxyphenyl)-N-phenylazetidin-2-imine 7ada

(MW = 358.4410 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6da



S16


eluent) to provide the corresponding azetidinimine with 80 % yield (57 mg, light brown solid).

1H NMR (300 MHz, CDCl3): 7.51 – 7.32 (m, 4H), 7.33 – 7.20 (m, 2H), 7.15 – 6.99 (m, 5H), 6.82 – 6.79 (m,

2H), 5.14 (dd, J = 6.0, 2.9 Hz, 1H), 3.75 (s, 3H), 3.50 (dd, J = 14.7, 6.0 Hz, 1H), 2.9 (dd, J = 14.7, 2.9 Hz,

1H).

13C NMR (75 MHz, CDCl3): δ 162.7 (d, J = 249.9 Hz, C), 155.1 (C), 153.9 (C), 148.7 (C), 135.3 (C), 133.7

(C), 129.1 (2CH), 127.8 (d, J = 8.3 Hz, 2CH), 123.1 (CH), 122.3 (2CH), 117.7 (2CH), 116.2 (d, J =21.7 Hz,

2CH), 114.4 (2CH), 57.7 (CH), 55.7 (CH3), 40.8 (CH2).

HRMS-ESI (m/z) calcd for C22H20FN2O [(M+H)+] 347.1481, found 347.1566.

IR (neat): 2926, 1720, 1609, 1365, 1217, 1134 cm-1.

m.p. 78.0 °C.

4-(4-iodophenyl)-1-(4-methoxyphenyl)-N-phenylazetidin-2-imine 7aea

(MW = 454.3115 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ea


2.0 equiv.) and SiO2 (12 mg, 0.2 mmol, 1.0 equiv.) in DMF (420 µL). The sealed tube was placed in a

microwave apparatus for 1 h at 100 °C. The crude material was purified by flash chromatography on

silica gel (10% ethyl acetate in heptane as eluent) to provide the corresponding azetidinimine with 61

% yield (54 mg, orange solid).

1H NMR (300 MHz, CDCl3): δ 7.72 (d, J = 7.8 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.33 – 7.28 (m, 3H), 7.18

(d, J = 7.5 Hz, 2H), 7.05 (d, J = 7.8 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 5.13 (dd, J = 3.0, 1.5 Hz, 1H), 3.77 (s,

3H), 3.52 (dd, J = 14.7, 3.0 Hz, 1H), 2.92 (dd, J = 14.7, 1.5 Hz, 1H).

S17

13C NMR (75 MHz, CDCl3): δ 154.0 (C), 152.7 (C), 147.3 (C), 138.2 (C), 137.2 (2CH), 132.4 (C), 128.0

(2CH), 126.9 (2CH), 122.0 (CH), 121.2 (2CH), 116.6 (2CH), 113.3 (2CH), 92.8 (C), 56.7 (CH), 54.5 (CH3),

39.4 (CH2).

HRMS-ESI (m/z) calcd for C22H19IN2O [M+H]+ 455.0620, found 455.0608.

IR (neat): 2951, 1674, 1510, 1487, 1389, 1241, 1160, 1010, 825, 698 cm-1.

mp: 139.1 °C.

4-(3,5-dichlorophenyl)-1-(4-methoxyphenyl)-N-phenylazetidin-2-imine 7aga

(MW = 397.2990 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ha



sealed tube was placed in a microwave apparatus for 1 h at 100 °C. The crude material was purified by


corresponding azetidinimine with 36 % yield (29 mg, yellow oil).

1H NMR (300 MHz, CDCl3): δ 7.43 – 7.06 (m, 7H), 7.02 – 6.86 (m, 3H), 6.84 – 6.69 (m, 2H), 5.38 (dd, J =

6.2, 3.0 Hz, 1H), 3.69 (s, 3H), 3.53 (dd, J = 14.7, 6.2 Hz, 1H), 2.76 (dd, J = 14.7, 3.0 Hz, 1H).

13C NMR (75 MHz, CDCl3): δ 155.2 (C), 153.6 (C), 148.3 (C), 135.4 (C), 134.5 (C), 133.5 (C), 133.1 (C),

129.8 (CH), 129.1 (2CH), 128.0 (CH), 127.9 (CH), 123.2 (CH), 122.2 (2CH), 117.6 (2CH), 114.5 (2CH), 55.7

(CH3), 54.9 (CH)., 39.4 (CH2).

HRMS-ESI (m/z) calcd for C22H19Cl2N2O [(M+H)+] 397.0796, found 397.0852.

IR (neat): 2901, 2853, 1670, 1597, 1542, 1527, 1511, 1418, 1366, 1305, 1184, 750 cm-1.

1-(4-methoxyphenyl)-4-(naphthalen-2-yl)-N-phenylazetidin-2-imine 7aha

S18

(MW = 378.4750 g.mol-1)






corresponding azetidinimine with 32 % yield (24 mg, white solid).

1H NMR (300 MHz, CDCl3): δ 7.94 – 7.76 (m, 4H), 7.62 – 7.38 (m, 5H), 7.35 – 7.25 (m, 3H), 7.05 (d, J =

9.1 Hz, 2H), 6.78 (d, J = 9.1 Hz, 2H), 5.31 (dd, J = 6.0, 2.9 Hz, 1H), 3.73 (s, 3H), 3.57 (dd, J = 14.7, 6.1 Hz,

1H), 3.02 (dd, J = 14.7, 2.9 Hz, 1H).


129.4 (CH), 129.1 (2CH), 128.1 (CH), 128.0 (CH), 126.7 (CH), 126.4 (CH), 125.5 (CH), 123.3 (CH), 123.0

(CH), 122.4 (2CH), 117.7 (2CH), 114.4 (2CH), 58.5 (CH), 55.6 (CH3), 40.6 (CH2).


IR (neat): 2932, 1742, 1592, 1510, 1440, 1338, 1245, 1160, 1094, 1032 cm-1.

m.p. 114.0 °C.

1-(4-methoxyphenyl)-N-phenyl-4-(pyridin-3-yl)azetidin-2-imine 7aia

(MW = 329.4030 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ia


S19




corresponding azetidinimine with 24 % yield (16 mg, brown oil).

1H NMR (300 MHz, CDCl3): δ 8.63 (br s, 1H), 8.53 (br s, 1H), 7.72 – 7.63 (m, 1H), 7.38 – 7.15 (m, 5H),

7.04 – 6.89 (m, 3H), 6.80 – 6.70 (m, 2H), 5.14 (dd, J = 6.1, 2.9 Hz, 1H), 3.68 (s, 3H), 3.49 (dd, J = 14.7,

6.1 Hz, 1H), 2.88 (dd, J = 14.7, 2.9 Hz, 1H).

13C NMR (75 MHz, CDCl3): δ 155.3 (C), 153.5 (C), 150.1 (CH), 148.4 (C), 148.3 (CH), 135.2 (C), 133.5 (CH),

133.4 (C), 129.2 (2CH), 124.2 (CH), 123.2 (CH), 122.3 (2CH), 117.7 (2CH), 114.5 (2CH), 55.9 (CH), 55.7

(CH3), 40.5 (CH2).


IR (neat): 2926, 1720, 1609, 1365, 1217, 1134 cm-1.

S20


N,4-bis(4-chlorophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7bba

(MW = 397.299 g.mol-1)


(49 mg, 0.2 mmol, 1.0 equiv.), ynamide 3b (100 mg, 0.4 mmol, 2.0 equiv.), t-BuOLi (182 µL, 0.4 mmol,



silica gel (10% ethyl acetate in heptane as eluent) to provide the corresponding azetidinimine with 29%

yield (29 mg, orange solid).

1H NMR (300 MHz, CDCl3): δ 7.37 – 7.23 (m, 8H), 6.94 (d, J = 8.7 Hz, 2H),6.81(d, J = 9.0 Hz, 2H), 5.15

(dd, J = 6.0, 3.0 Hz, 1H), 3.76 (s, 3H), 3.50 (dd, J = 14.7, 6.0 Hz, 1H), 2.89 (dd, J = 14.7, 3.0 Hz, 1H).


129.3 (2CH), 128.9 (C), 127.2 (2CH), 123.4 (2CH), 117.5 (2CH), 114.2 (2CH), 57.5 (CH), 55.5 (CH3), 40.3

(CH2).

HRMS-ESI (m/z) calcd for C22H1935Cl2N2O [M+H]+ 398.0874, found 398.0898.

IR (neat): 2954, 1672, 1511, 1487, 1388, 1241, 1160, 1090, 826 cm-1.

4-(4-chlorophenyl)-N-(4-bromophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7cba

(MW = 441.753 g.mol-1)

S21


(49 mg, 0.2 mmol, 1.0 equiv.), ynamide 3c (118 mg, 0.4 mmol, 2.0 equiv.), t-BuOLi (182 µL, 0.4 mmol,




% yield (35.1 mg, orange solid).

1H NMR (300 MHz, CDCl3): δ 7.32 – 7.24 (m, 8H), 6.82 (d, J = 8.7 Hz, 2H), 6.73 (d, J = 8.7 Hz, 2H), 5.08


13C NMR (75 MHz, CDCl3): δ 154.0 (C), 153.8 (C), 145.3 (C), 137.4 (C), 131.9 (2CH), 130.4 (C), 129.3

(2CH), 129.0 (C), 127.3 (2CH), 123.9 (2CH), 117.2 (2CH), 114.3 (2CH), 100.2 (C), 57.6 (CH), 55.5 (CH2),

40.3 (CH2).

HRMS-ESI (m/z) calcd for C22H1979Br35ClN2O [M+H]+ 441.0369, found 441.0375; (m/z) calcd for

C22H2081Br35ClN2O [M+H]+ 443.0343, found 443.0351.

IR (neat): 2953, 1673, 1579, 1511, 1484, 1242, 1161, 827 cm-1.

4-(4-chlorophenyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dba

(MW = 488.7535 g.mol-1)


(49 mg, 0.2 mmol, 1.0 equiv.), ynamide 3d (137 mg, 0.4 mmol, 2.0 equiv.), t-BuOLi (182 µL, 0.4 mmol,





S22

1H NMR (300 MHz, CDCl3): δ 7.57 (d, J = 8.4 Hz, 2H), 7.36 – 7.33 (m, 6H), 6.82 (d, J = 7.2 Hz, 2H), 6.78

(d, J = 8.4 Hz, 2H), 5.14 (dd, J = 6.1, 2.9 Hz, 1H), 3.76 (s, 3H), 3.49 (dd, J = 14.6, 6.1 Hz, 1H), 2.87 (dd, J =

14.6, 2.9 Hz, 1H).

13C NMR (75 MHz, CDCl3): δ 155.1 (C), 153.8 (C), 148.2 (C), 137.9 (2CH), 137.6 (C), 134.2 (C), 133.1 (C),

129.3 (2CH), 127.2 (2CH), 124.4 (2CH), 117.5 (2CH), 114.2 (2CH), 86.2 (C), 57.3 (CH), 55.5 (CH3), 40.3

(CH2).

HRMS-ESI (m/z) calcd for C22H1935ClIN2O [M+H]+ 489.0230, found 489.0240.

IR (neat): 2952, 1668, 1510, 1480, 1387, 1241, 1160, 825 cm-1.

4-(4-chlorophenyl)-N-(4-trifluoromethylphenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7eba

(MW = 430.8552 g.mol-1)


(49 mg, 0.2 mmol, 1.0 equiv.), ynamide 3e (114 mg, 0.4 mmol, 2.0 equiv.), t-BuOLi (182 µL, 0.4 mmol,





1H NMR (300 MHz, CDCl3): δ 7.42 – 7.27 (m, 8H), 7.18 (d, J = 7.8 Hz, 2H), 6.82 (d, J = 9.0 Hz, 2H), 5.17


13C NMR (75 MHz, CDCl3): δ 155.2 (C), 154.3 (C), 149.0 (C), 137.4 (C), 134.2 (C), 133.0 (C), 131.4 (q, J =

32.4 Hz, CCF3), 129.4 (2CH), 127.2 (2CH), 125.3 (2CH), 119.3 (q, J = 4 Hz, CH), 119.1 (q, J = 4 Hz, CH),

117.6 (2CH), 114.3 (2CH), 57.5 (CH), 55.4 (CH3), 40.3 (CH2). The signal for CF3 was not observed.

19F NMR (300 MHz, C6D6): δ – 62.2 (s).

HRMS-ESI (m/z) calcd for C23H2935ClF3N2O [M+H]+ 431.1138, found 431.1131.

S23

IR (neat): 2931, 1676, 1512, 1329, 1243, 1162, 1123, 827 cm-1.

4-(4-chlorophenyl)-N-(4-methoxyphenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7fba

(MW = 392.8830 g.mol-1)


(49 mg, 0.2 mmol, 1.0 equiv.), ynamide 3f (98 mg, 0.4 mmol, 2.0 equiv.), t-BuOLi (182 µL, 0.4 mmol,

2.0 equiv.) and SiO2 (12 mg, 0.2 mmol, 1.0 equiv.) in DMF (420 µL)The sealed tube was placed in a



% yield (26 mg, brown solid).

1H NMR (300 MHz, CDCl3): δ 7.37 (m, 6H), 6.98 (d, J = 8.7 Hz, 2H), 6.87 – 7.80 (m, 4H), 5.13 (dd, J = 6.0,

3.0 Hz, 1H), 3.80 (s, 3H), 3.76 (s, 3H), 3.53 (dd, J = 14.7, 6.0 Hz, 1H), 2.92 (dd, J = 14.7, 3.0 Hz, 1H).


127.3 (2CH), 122.9 (2CH), 117.4 (2CH), 114.2 (4CH), 57.5 (CH), 55.4 (2CH3), 40.6 (CH2). One Cq was not

observed.

HRMS-ESI (m/z) calcd for C23H2235ClN2O2 [M+H]+ 393.1373, found 393.1370.

IR (neat): 2954, 2838, 1677, 1503, 1387, 1238, 1160, 1035, 828 cm-1.

4-(2,4-dichlorophenyl)-N-(4-bromophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7cfa

S24

(MW = 476.1927g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6fa






1H NMR (300 MHz, CDCl3): δ 7.45 (d, J = 2.1 Hz, 1 H), 7.40 (d, J = 1.8 Hz, 1 H), 7.37 (d, J = 2.4 Hz, 1 H),

7.32 – 7.22 (m, 4 H), 6.91 (d, J = 9.0 Hz, 2 H), 6.87 (d, J = 9.0 Hz, 2 H), 5.52 (dd, J = 6.0, 3.0 Hz, 1 H), 3.79

(s, 3H), 3.62 (dd, J = 14.7, 6.0 Hz, 1H), 2.86 (dd, J = 14.7, 3.0 Hz, 1 H).

13C NMR (75 MHz, CDCl3): δ 152.3 (C), 131.9 (2CH), 131.8 (CH), 130.4 (C), 129.6 (CH), 127.6 (2CH), 124.5

(C), 123.3 (2CH), 117.8 (CH), 114.4 (2CH), 102.2 (C), 99.9 (C), 55.5 (CH), 55.0 (CH3), 39.0 (CH2).

HRMS-ESI (m/z) calcd for C22H1881Br35Cl2N2O [M+H]+ 476.9959, found 476.9955.

IR (neat): 2932, 2835, 1674, 1510, 1483, 1390, 1242, 1161, 826 cm-1.

4-(2,4-dichlorophenyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dfa

(MW = 521.9763 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6fa



S25




1H NMR (300 MHz, CDCl3): δ 7.56 (d, J = 8.7 Hz, 2 H), 7.45 (d, J = 1.8 Hz, 1 H), 7.36 (d, J = 9.0 Hz, 2 H), 7.29

(d, J = 8.4 Hz, 1 H), 7.23 (d, J = 1.8 Hz, 1 H), 6.86 (d, J = 9.0 Hz, 2 H), 6.75 (d, J = 8.7 Hz, 2 H), 5.48 (dd, J = 6.3,

3.0 Hz, 1 H), 3.78 (s, 3H), 3.60 (dd, J = 14.7, 6.3 Hz, 1H), 2.84 (dd, J = 14.7, 3.0 Hz, 1 H).

13C NMR (75 MHz, CDCl3): δ 155.2 (C), 153.7 (C), 148.0 (C), 137.9 (2CH), 135.0 (C), 134.4 (C), 133.1 (C),

133.0 (C), 129.6 (CH), 127.8 (CH), 127.7 (CH), 124.3 (2CH), 117.5 (2CH), 114.4 (2CH), 86.4 (C), 55.5 (CH),

54.7 (CH3), 39.1 (CH2)

HRMS-ESI (m/z) calcd for C22H1835Cl2IN2O [M+H]+ 522.9836, found 522.9842.

IR (neat): 2953, 2833, 1667, 1509, 1388, 1240, 1161, 1045, 1035, 825, 731 cm-1.

4-(2,4-dichlorophenyl)-N-(4-trifluoromethylphenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7efa

(MW = 430.8552 g.mol-1)


(49 mg, 0.2 mmol, 1.0 equiv.), ynamide 3e (114 mg, 0.4 mmol, 2.0 equiv.), t-BuOLi (182 µL, 0.4 mmol,





1H NMR (300 MHz, CDCl3): δ 7.53 (d, 2H, J = 8.1 Hz), 7.46 (d, 1H, J = 1.8 Hz), 7.37 (d, 2H, J = 8.7 Hz),

7.32 (d, 1H, J = 8.1 Hz), 7.24 (d, 1H, J = 8.1 Hz), 7.06 (d, 2H, J = 8.1 Hz), 6.87 (d, 2H, J = 8.7 Hz), 5.53 (dd,

J = 5.4, 2.4 Hz, 1 H), 3.79 (s, 3H), 3.62 (dd, J = 14.7, 5.4 Hz, 1H), 2.85 (dd, J = 14.7, 2.4 Hz, 1 H).

S26


127.7 (2CH), 126.1 (CH), 122.2 (2CH), 117.7 (2CH), 114.4 (3CH), 55.5 (CH3), 54.9 (CH), 39.1 (CH2). The

signals for CCF3 and CF3 were not observed.

HRMS-ESI (m/z) calcd for C23H2035ClF3N2O [M+H]+ 465.0743, found 465.0754.

IR (neat): 2905, 2835, 1678, 1605, 11508, 1323, 1244, 1162, 1065, 826 cm-1.

S27


4-(naphtyl)-N-(4-chlorophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7bha

(MW = 412.9105 g.mol-1)







1H NMR (300 MHz, CDCl3): δ 7.88 – 7.84 (m, 5 H), 7.53 – 7.50 (m, 2 H), 7.42 (d, J = 8.7 Hz, 2 H), 7.23 (d,

J = 1.8 Hz, 2 H), 6.99 (d, J = 8.4 Hz, 2 H), 6.80 (d, J = 9.3 Hz, 2 H), 5.33 (dd, J = 6.0, 3.0 Hz, 1 H), 3.74 (s,

3H), 3.56 (dd, J = 14.7, 6.0 Hz, 1H), 3.00 (dd, J = 14.7, 3.0 Hz, 1 H);

13C NMR (75 MHz, CDCl3): δ 154.4 (C), 152.1(C), 147.1 (C), 136.5 (C), 133.5 (C), 133.3 (2C), 129.2 (CH),

129.0 (C), 128.9 (2CH), 127.9 (CH), 127.8 (CH) 126.5 (CH), 126.3 (CH), 125.3 (CH), 123.4 (2CH), 123.0

(CH), 117.6 (2CH), 114.1 (2CH), 58.4 (CH3), 55.4 (CH), 40.3 (CH2).

HRMS-ESI (m/z) calcd for C26H2235ClN2O [(M+H)+] 413.1416, found 413.1421.

IR (neat): 1989, 1670, 1586, 1511, 1487, 1391, 1242, 1161, 1036, 827, 747 cm-1.

4-(naphtyl)-N-(4-bromophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7cha

(MW = 457.3618 g.mol-1)

S28






yield (18 mg, brown solid).

1H NMR (300 MHz, C6D6): δ 7.63 (d, J = 9.3 Hz, 2 H), 7.54 (d, J = 2.4 Hz, 2 H), 7.33 (d, J = 8.7 Hz, 2 H),

7.22 – 7.19 (m, 5 H), 6.84 (d, J = 8.7 Hz, 2 H), 6.69 (d, J = 9.0 Hz, 2 H), 4.63 (dd, J = 6.0, 3.0 Hz, 1 H), 3.21

(s, 3H), 2.81 (dd, J = 14.7, 6.0 Hz, 1H), 2.45 (dd, J = 14.7, 3.0 Hz, 1 H);

13C NMR (75 MHz, C6D6): δ 155.5 (C), 150.1 (C), 139.5 (C), 135.7 (C), 133.7 (C), 133.6 (C), 132.2 (2CH),

132.0 (C), 129.3 (CH), 127.4, 126.6 (CH), 126.3 (CH), 125.3 (CH), 124.3 (2CH), 123.2 (CH), 117.9 (2CH),

114.4 (2CH), 72.2 (C), 58.4 (CH3), 54.7 (CH), 40.1 (CH2). Due to overlap with the solvent peak some

signals could not be observed.

HRMS-ESI (m/z) calcd for C26H2279BrN2O [(M+H)+] 457.0911, found 457.0915.

IR (neat): 2951, 2834, 1670, 1510, 1484, 1242, 1161, 1070, 1035, 1006, 826, 749 cm-1.

4-(naphtyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dha

(MW = 504.3623 g.mol-1)






yield (37 mg, brown solid).

S29

1H NMR (300 MHz, C6D6): δ 7.88 – 7.82 (m, 5 H), 7.59 (d, J = 8.7 Hz, 2 H), 7.52 (d, J = 8.7 Hz, 2 H), 7.42

(d, J = 8.7 Hz, 2 H), 6.82 (d, J = 8.7 Hz, 2 H), 6.79 (d, J = 8.7 Hz, 2 H), 5.33 (dd, J = 6.0, 3.0 Hz, 1 H), 3.73

(s, 3H), 3.56 (dd, J = 14.7, 6.0 Hz, 1H), 3.00 (dd, J = 14.7, 3.0 Hz, 1 H);

13C NMR (75 MHz, C6D6): δ 155.0 (C), 154.1 (C), 147.5 (C), 142.2 (C), 137.8 (2CH), 136.5 (C), 133.5 (C),

133.3 (CH), 129.3 (CH), 127.9 (CH), 127.8 (CH), 126.5 (CH), 126.3 (CH), 125.3 (CH), 124.5 (2CH), 123.0

(CH), 117.6 (2CH), 114.2 (2CH), 90.2 (C), 58.4 (CH3), 55.4 (CH), 40.3 (CH2).

HRMS-ESI (m/z) calcd for C26H22IN2O [(M+H)+] 505.0772, found 505.0793.

IR (neat): 2990, 1669, 1575, 1510, 1480, 1386, 1242, 1161, 1036, 828, 748 cm-1.

1-(4-ethoxyphenyl)-4-(naphthalen-2-yl)-N-phenylazetidin-2-imine 7ahb

(MW = 392.5020 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 7hb





corresponding azetidinimine with 26% yield (20 mg, yellow solid).

1H NMR (300 MHz, CDCl3): δ 7.94 – 7.78 (m, 4H), 7.62 – 7.37 (m, 5H), 7.36 – 7.24 (m, 2H), 7.10 – 6.98

(m, 3H), 6.84 – 6.71 (m, 2H), 5.31 (dd, J = 6.0, 2.9 Hz, 1H), 3.94 (q, J = 7.0 Hz, 2H), 3.57 (dd, J = 14.7, 6.0

Hz, 1H), 3.01 (dd, J = 14.7, 2.9 Hz, 1H), 1.35 (t, J = 7.0 Hz, 3H).


129.4 (CH), 129.1 (2CH), 128.0 (CH), 127.9 (CH), 126.7 (CH), 126.4 (CH), 125.5 (CH), 123.3 (CH), 123.0

(CH), 122.4 (2CH), 117.6 (2CH), 115.0 (2CH), 63.8 (CH2), 58.5 (CH), 40.6 (CH2), 15.0 (CH3).

HRMS-ESI (m/z) calcd for C27H25N2O [(M+H)+] 393.1967, found 393,1952.

S30

IR (neat): 3048, 2978, 1734, 1673, 1591, 1497, 1488, 1477, 1444, 1391, 1296, 1239, 1195, 1160, 1047,

922 cm-1.

m.p. 114.0 °C.

1-(4-ethoxyphenyl)-4-(naphthalen-2-yl)-N-phenylazetidin-2-imine 7bhb

(MW = 392.5020 g.mol-1)






yield (31 mg, yellow solid).


J = 9.9 Hz, 1 H), 7.07 (d, J = 8.7 Hz, 2 H), 6.86 (d, J = 9.0 Hz, 2 H), 5.42 (dd, J = 6.0, 2.7 Hz, 1 H), 4.04 (q, J

= 6.9 Hz, 2H), 3.64 (dd, J = 14.7, 6.0 Hz, 1H), 3.08 (dd, J = 14.7, 2.7 Hz, 1 H), 1.44 (t, J = 6.9 Hz, 3H).

13C NMR (75 MHz, CDCl3): δ 154.3 (C), 136.6 (C), 133.3 (C), 129.2 (CH), 128.9 (2CH), 127.9 (CH), 127.8

(CH), 126.5 (CH), 126.3 (CH), 125.3 (CH), 123.4 (2CH), 123.0 (CH), 117.5 (2CH), 114.9 (2CH), 63.6 (CH2),

58.4 (CH), 40.3 (CH2), 14.8 (CH3).

HRMS-ESI (m/z) calcd for C27H2435ClN2O [(M+H)+] 427.1572, found 427.1577.

IR (neat): 2980, 1672, 1586, 1510, 1487, 1392, 1239, 1161, 1090, 1047, 836, 748 cm-1.

4-(naphtyl)-N-(4-iodophenyl)-1-(4-ethoxyphenyl)azetidin-2-imine 7dhb

S31

(MW = 392.5020 g.mol-1)








J = 9.9 Hz, 1 H), 7.07 (d, J = 8.7 Hz, 2 H), 6.86 (d, J = 9.0 Hz, 2 H), 5.42 (dd, J = 6.0, 2.7 Hz, 1 H), 4.04 (q, J

= 6.9 Hz, 2H), 3.64 (dd, J = 14.7, 6.0 Hz, 1H), 3.08 (dd, J = 14.7, 2.7 Hz, 1 H), 1.44 (t, J = 6.9 Hz, 3H).

13C NMR (75 MHz, CDCl3): δ 154.4 (C), 154.3 (C), 148.4 (C), 137.8 (2CH), 136.5 (2C), 133.4 (CH), 129.2

(CH), 127.9 (CH), 127.8 (CH), 126.5 (CH), 126.3 (CH), 125.3 (CH), 124.5 (2CH), 123.0 (CH), 117.6 (2CH),

114.9 (2CH), 86.1 (C), 63.6 (CH2), 58.4 (CH), 40.3 (CH2), 14.6 (CH3)

HRMS-ESI (m/z) calcd for C27H24IN2O [(M+H)+] 519.0928, found 427.1577.

IR (neat): 2977, 2926, 1666, 1509, 1478, 1389, 1238, 1161, 833, 819, 747, 732cm-1.

4-(naphtyl)-N-(4-methoxphenyl)-1-(4-ethoxyphenyl)azetidin-2-imine 7fhb

(MW = 422.5183 g.mol-1)


(57 mg, 0.2 mmol, 1.0 equiv.), ynamide 3f (136 mg, 0.4 mmol, 2.0 equiv.), t-BuOLi (182 µL, 0.4 mmol,


S32




1H NMR (300 MHz, CDCl3): δ 7.89 – 7.83 (m, 2 H), 7.56 (d, J = 1.8 Hz, 2 H), 7.53 – 7.46 (m, 3 H), 7.44 (d,

J = 4.5 Hz, 2 H), 7.02 (d, J = 9.0 Hz, 2 H), 6.87 (d, J = 9.0 Hz, 2 H), 6.79 (d, J = 9.0 Hz, 2 H), 5.30 (dd, J =

6.0, 3.0 Hz, 1 H), 3.95 (q, J = 6.9 Hz, 2H), 3.80 (s, 3H), 3.58 (dd, J = 14.7, 6.0 Hz, 1H), 3.02 (dd, J = 14.7,

3.0 Hz, 1 H), 1.37 (t, J = 6.9 Hz, 3H).


133.3 (C), 129.2 (C), 127.9 (C), 127.8 (C), 126.5 (C), 126.2 (C), 125.3 (C), 123.2 (C), 123.0 (2CH), 117.4

(2CH), 114.9 (2CH), 114.2 (2CH), 63.6 (CH2), 58.4 (CH), 55.4 (CH3), 40.6 (CH2), 14.8 (CH3).


IR (neat): 2978, 2833, 1669, 1501, 1389, 1235, 1160, 1037, 834, 820, 749, 732 cm-1.

S33


4-(4-fluorophenyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dda

(MW = 472.2941 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6da




silica gel (10% ethyl acetate in heptane as eluent) to provide the corresponding azetidinimine with 7 %


1H NMR (300 MHz, C6D6): δ 7.54 – 7.49 (m, 4 H), 6.75 – 6.67 (m, 8 H), 4.30 (dd, J = 6.0, 3.0 Hz, 1 H), 3.24

(s, 3H), 2.67 (dd, J = 14.7, 6.0 Hz, 1H), 2.20 (dd, J = 14.7, 3.0 Hz, 1 H).

13C NMR (75 MHz, C6D6): δ 153.7 (C), 140.2 (C), 138.3 (C), 138.2 (d, J = 5.0 Hz, 2CH), 131.9 (C), 131.5

(C), 124.7(2CH), 117.8 (2CH), 115.9 (d, J = 21.3 Hz, 2CH), 114.4(2CH), 86.2 (C), 57.4 (CH), 54.8 (CH3),

40.2 (CH2). Due to overlap with the solvent peak some signals could not be observed.

IR (neat): 2970, 2926, 1739, 1693, 1599, 1584, 1449, 1229, 1207, 1110, 1015 cm-1.

4-(4-chlorophenyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dea

(MW = 580.2001 g.mol-1)

S34

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ea






1H NMR (300 MHz, CDCl3): δ 7.63 (d, J = 8.4 Hz, 2 H), 7.49 (d, J = 8.7 Hz, 2 H), 7.28 (d, J = 8.7 Hz, 2 H),

7.07 (d, J = 8.4 Hz, 2 H), 6.75-6.72 (m, 4 H), 5.06 (dd, J = 3.0, 1.5 Hz, 1 H), 3.67 (s, 3H), 3.43 (dd, J = 14.7,

3.0 Hz, 1H), 2.81 (dd, J = 14.7, 1.5 Hz, 1 H).

13C NMR (75 MHz, CDCl3): δ 138.2 (2CH), 137.9 (2CH), 127.6 (2CH), 124.4 (2CH), 117.8 (2CH), 114.3

(2CH), 99.9 (2C), 55.5 (CH+CH3), 40.1 (CH2).

HRMS-ESI (m/z) calcd for C22H19I2N2O [M+H]+ 580.9582, found 580.9583.

IR (neat): 2931, 2832, 1667, 1509, 1481, 1240, 1160, 1004, 836, 731 cm-1.

4-(3,5-dichlorophenyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dga

(MW = 521.9763 g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6ga






1H NMR (300 MHz, C6D6): δ 7.51 (d, J = 8.7 Hz, 2 H), 7.41 (d, J = 9.3 Hz, 2 H), 6.96– 6.91 (m, 3 H), 6.67

(d, J = 9.0 Hz, 2 H), 6.62 (d, J = 9.3 Hz, 2 H), 4.11 (dd, J = 6.0, 2.7 Hz, 1 H), 3.22 (s, 3H), 2.56 (dd, J = 14.4,

6.0 Hz, 1H), 2.09 (dd, J = 14.4, 2.7 Hz, 1 H).

S35

13C NMR (75 MHz, C6D6): δ 153.6 (C), 151.5 (C), 143.2 (C), 138.2 (2CH), 135.9 (2C), 128.6 (CH), 124.7

(2CH), 124.3 (2CH), 117.6 (2CH), 114.5 (2CH), 88,2 (C), 56.9 (CH), 54.7 (CH3), 39.8 (CH2).


IR (neat): 2931, 2834, 1677, 1591, 1511, 1243, 1161, 1035, 798, 699 cm-1.

4-(2,4-dichlorophenyl)-N-(4-iodophenyl)-1-(4-benzyloxyphenyl)azetidin-2-imine 7dfc

(MW = 598.0075g.mol-1)

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6fc






1H NMR (300 MHz, C6D6): δ .7.53 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.8 Hz, 2H), 7.23 (d, J = 7.5 Hz, 2H),

7.14-7.04 (m, 4H), 6.87-6.81 (m, 3H), 6.68 (d, J = 8.7 Hz, 1H), 6.64 (d, J = 8.3 Hz, 2H), 4.78 (dd, J = 6.1,

2.8 Hz, 1H, CH), 4.69 (s, 2H, OCH2), 2.75 (dd, J = 14.7, 6.1 Hz, 1H, CH2), 2.10 (dd, J = 14.7, 2.8 Hz, 1H,

CH2).

13C NMR (75 MHz, C6D6): δ 154.8 (C), 153.4 (C), 148.5 (C), 138.1 (2CH), 137.8 (CH), 137.4 (CH), 135.3

(C), 134.2 (C), 133.9 (C), 132.9 (C), 129.5 (CH), 128.5 (2CH), 127.7 (2CH), 124.7 (2CH), 120.2 (C), 117.7

(2CH), 115.6 (2CH), 86.5 (C), 70.2 (CH2), 54.8 (CH), 36.9 (CH2). Due to overlap with the solvent peak

some signals could not be observed.


IR (neat): 3063, 2923, 2864, 1666, 1575, 1510, 1384, 1233, 1160, 1035, 822 cm-1

S36

4-(2,4-dichlorophenyl)-N-(4-iodophenyl)-1-(4-hydroxyphenyl)azetidin-2-imine 7dfm

(MW = 507.9606 g.mol-1)

To a solution of 7dfc (300 mg, 0.50 mmol, 1.0 equiv) in DCM (80 mL) at 0 °C was added AlCl3 (400 mg,

3.0 mmol, 6.0 equiv) followed by anisole (0.54 mL, 5.0 mmol, 10 equiv). The reaction mixture was

stirred at 0 °C for 1 h before being quenched with aqueous HCl (1N). The organic layer was extracted

with DCM, washed with sat. NaHCO3 then brine, dried over MgSO4, filtered and concentrated in vacuo.

Pure 7dfm was obtained after purification by flash column chromatography (0-40% EtOAc in Petroleum

Ether) as an orange oil (210 mg, 0.41 mmol, 82%).

1H NMR (300 MHz, C6D6): δ 7.49 (d, J = 8.7 Hz, 2 H), 7.33 (d, J = 9.0 Hz, 2 H), 7.08 (d, J = 2.1 Hz, 1 H),

6.79 (d, J = 9.3 Hz, 2 H), 6.67-6.65 (m, 1 H), 6.60 (d, J = 8.7 Hz, 1 H), 6.49 (d, J = 9.0 Hz, 2 H), 4.75 (dd, J

= 6.3, 3.0 Hz, 1 H), 2.72 (dd, J = 14.7, 6.3 Hz, 1H), 2.08 (dd, J = 14.7, 3.0 Hz, 1 H).

13C NMR (75 MHz, C6D6): δ 153.5 (C), 151.8 (C), 148.5 (C), 138.2 (2CH), 135.3 (C), 134.2 (C), 132.8 (C),

127.6 (CH), 124.7 (2CH), 117.8 (2CH), 115.7 (2CH), 86.5 (C), 54.7 (CH), 38.8 (CH2). Due to overlap with

the solvent peak some signals could not be observed.


IR (neat): 3318, 2924, 2853, 1662, 1576, 1520, 1479, 1387, 1234, 1161, 1035, 829 cm-1

(4-(2-(2,4-dichlorophenyl)-4-((4-iodophenyl)imino)azetidin-1-yl)phenyl)methanol 7dfn

(MW = 523.1955 g.mol-1)

S37

Prepared according to the general procedure for the formation of the azetidinimines, with: imine 6fn


2.0 equiv.) in DMF (420 µL). The sealed tube was placed in a microwave apparatus for 1 h at 100 °C.

The crude material was purified by flash chromatography on silica gel (0-50% ethyl acetate in heptane

as eluent) to provide the corresponding azetidinimine with 64 % yield (67 mg, orange solid).

1H NMR (300 MHz, C6D6): δ 7.54-7.46 (m, 4H), 7.16-7.14 (m, 2H), 7.11 (d, J = 2.0 Hz, 1H), 6.81 (d, J = 8.4

Hz, 1H), 6.68 (dd, J = 8.4, 2.0 Hz, 1H), 6.64-6.59 (m, 2H), 4.81 (dd, J = 6.3, 3.0 Hz, 1H), 4.25 (s, 2H), 2.72

(dd, J = 14.8, 6.3 Hz, 1H), 2.08 (dd, J = 14.8, 3.0 Hz, 1H).

13C NMR (75 MHz, C6D6): δ 153.9 (C), 148.5 (C), 139.3 (C), 138.4 (2CH), 136.3 (C), 135.3 (C), 134.5 (C),

133.1 (C), 129.8 (CH), 128.4 (2CH), 128.0 (CH), 127.9 (CH), 124.8 (2CH), 116.7 (2CH), 86.9 (C), 64.7 (CH2),

55.0 (CH), 39.0 (CH2).

HRMS-ESI (m/z) calcd for C22H18N2OI35Cl2 [M+H]+: 522.9835, found 522.9811.

IR (neat): 3363, 2921, 2853, 1667, 1576, 1509, 1480, 1387, 1237, 1160, 1073, 820 cm-1

4-(2-(2,4-dichlorophenyl)-4-((4-iodophenyl)imino)azetidin-1-yl)benzoic acid 7dfo

(MW = 537.1785 g.mol-1)

To a solution of 7dfn (0.039 mmol, 19 mg, 1.0 equiv) in acetone (0.9 mL) was added KMnO4 (0.073

mmol, 12 mg, 2.0 equiv). The reaction mixture was stirred at rt for 4 h before being quenched by

addition of a saturated aqueous solution of Na2SO3 (0.6 mL). After 5 min stirring at rt, an aqueous

solution of HCl (2 N, 1.2 mL) was added and the reaction was stirred until it became colorless. Organics

were extracted with DCM, combined, dried over MgSO4, filtered and concentrated in vacuo.

Purification by preparative TLC (5% MeOH in DCM) afforded the pure desired product as a white solid

(3 mg, 0.005 mmol, 13%). The small amount of material available did not allow a 13C NMR and IR

spectrum from being obtained.

S38

1H NMR (500 MHz, (CD3)2CO): δ 8.0 (d, J = 8.5 Hz, 2H), 7.64 (d, J = 8.3 Hz, 2H), 7.61 (s, 1H), 7.56 (d, J =

8.4 Hz, 2H), 7.42 (s, 2H), 6.92 (d, J = 8.4 Hz, 2H), 5.77 (dd, J = 6.3, 2.8 Hz, 1H), 3.88 (dd, J = 14.9, 6.3 Hz,

1H), 3.09 (dd, J = 14.9, 2.8 Hz, 1H)

HRMS-ESI (m/z) calcd for C22H16N2O2I35Cl2 [M+H]+: 536.9628, found 536.9622.

S39

8. NMR Spectra of Azetidinimines in Table 1

1-(4-methoxyphenyl)-N-4-diphenylazetidin-2-imine 7aaa

S40

1-(4-methoxyphenyl)-N-4-diphenylazetidin-2-imine 7aab

S41

1-(4-methoxyphenyl)-N-4-diphenylazetidin-2-imine 7aac

S42

1-(3-methoxyphenyl)-N-4-diphenylazetidin-2-imine 7aad

S43

S44

1-(3,4-dimethoxyphenyl)-N-4-diphenylazetidin-2-imine 7aaf

S45

1-(benzo[d][1,3]dioxol-5-yl)-N,4-diphenylazetidin-2-imine 7aag

S46

1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-N-4-diphenylazetidin-2-imine 7aah

S47

N,4-diphenyl-1-(3,4,5-trimethoxyphenyl)azetidin-2-imine 7aai

S48

HMQC of 7aai

Superimposition of the HMQC (purple) and the HMBC (green) of 7aai

S49

1-phenyl-N-4-diphenylazetidin-2-imine 7aaj

S50

1-(4-iodophenyl)-N-4-diphenylazetidin-2-imine 7aak

S51

1-(4-(methylthio)phenyl)-N,4-diphenylazetidin-2-imine 7aal

S52

(E)-4-(2-phenyl-4-(phenylimino)azetidin-1-yl)phenol 7aam

S53


4-(4-chlorophenyl)-1-(4-methoxyphenyl)-N-phenylazetidin-2-imine 7aba

S54

1,4-bis(4-methoxyphenyl)-N-phenylazetidin-2-imine 7aca

S55

1,4-bis(4-methoxyphenyl)-N-phenylazetidin-2-imine 7ada

S56

4-(4-iodophenyl)-1-(4-methoxyphenyl)-N-phenylazetidin-2-imine 7aea

S57

4-(3,5-dichlorophenyl)-1-(4-methoxyphenyl)-N-phenylazetidin-2-imine 7aga

S58

1-(4-methoxyphenyl)-4-(naphthalen-2-yl)-N-phenylazetidin-2-imine 7aha

S59

1-(4-methoxyphenyl)-N-phenyl-4-(pyridin-3-yl)azetidin-2-imine 7aia

S60


N,4-bis(4-chlorophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7bba

S61

4-(4-chlorophenyl)-N-(4-bromophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7cba

S62

4-(4-chlorophenyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dba

S63

4-(4-chlorophenyl)-N-(4-trifluoromethylphenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7eba

S64

4-(4-chlorophenyl)-N-(4-methoxyphenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7fba

S65

4-(2,4-dichlorophenyl)-N-(4-bromophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7cfa

S66

4-(2,4-dichlorophenyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dfa

S67

4-(2,4-dichlorophenyl)-N-(4-trifluoromethylphenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7efa

S68


4-(naphtyl)-N-(4-chlorophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7bha

S69

4-(naphtyl)-N-(4-bromophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7cha

S70

4-(naphtyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dha

S71


S72


S73

4-(naphtyl)-N-(4-chlorophenyl)-1-(4-ethoxyphenyl)azetidin-2-imine 7bhb

S74

4-(naphtyl)-N-(4-iodophenyl)-1-(4-ethoxyphenyl)azetidin-2-imine 7dhb

S75

4-(naphtyl)-N-(4-methoxphenyl)-1-(4-ethoxyphenyl)azetidin-2-imine 7fhb

S76


4-(4-fluorophenyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dda

S77

4-(4-iodophenyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dea

S78

4-(3,5-dichlorophenyl)-N-(4-iodophenyl)-1-(4-methoxyphenyl)azetidin-2-imine 7dga

S79

4-(2,4-dichlorophenyl)-N-(4-iodophenyl)-1-(4-benzyloxyphenyl)azetidin-2-imine 7dfc

S80

4-(2,4-dichlorophenyl)-N-(4-iodophenyl)-1-(4-hydroxyphenyl)azetidin-2-imine 7dfm

S81

(4-(2-(2,4-dichlorophenyl)-4-((4-iodophenyl)imino)azetidin-1-yl)phenyl)methanol 7dfn

S82

4-(2-(2,4-dichlorophenyl)-4-((4-iodophenyl)imino)azetidin-1-yl)benzoic acid 7dfo

download fileview on ChemRxivSupplementaryAZETI-ChemRxiv.pdf (4.49 MiB)



Documents

Azetidinimines as a Novel Series of Non-Covalent Broad