Design of gp120 HIV-1 entry inhibitors by scaffold hopping via isosteric replacements

European Journal of Medicinal Chemistry 224 (2021) 1 13681

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Design of gp120 HIV-1 entry inhibitors by scaffold hopping via

isosteric replacements

Ildar R. Iusupov ^a, Francesca Curreli ^b, Evgeniy A. Spiridonov ^a, Pavel O. Markov ^a,

,

*

Shahad Ahmed ^b, Dmitry S. Belov ^a, Ekaterina V. Manasova ^a, Andrea Altieri ^a

,

, ***

Alexander V. Kurkin ^a^**, Asim K. Debnath ^b

^aEDASA Scientiﬁc, Scientiﬁc Campus, Moscow State University, Leninskie Gory Bld. 75, 77-101b, Moscow, 119992, Russia

^bLaboratory of Molecular Modeling & Drug Design, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E 67th Street, New York, 10065, New

York, United States

a r t i c l e i n f o

a b s t r a c t

Article history:

We present the development of alternative scaffolds and validation of their synthetic pathways as a tool

for the exploration of new HIV gp120 inhibitors based on the recently discovered inhibitor of this class,

NBD-14136. The new synthetic routes were based on isosteric replacements of the amine and acid

precursors required for the synthesis of NBD-14136, guided by molecular modeling and chemical

feasibility analysis. To ensure that these synthetic tools and new scaffolds had the potential for further

exploration, we eventually tested few representative compounds from each newly designed scaffold

against the gp120 inhibition assay and cell viability assays.

Received 8 February 2021

Received in revised form

28 June 2021

Accepted 29 June 2021

Available online xxx

Keywords:

HIV

gp120

Drug discovery

Lead optimization

Synthesis

Screening

1. Introduction

identiﬁed [1e3] and validated as critical players in the AIDS pa-

thology. Among the HIV entry targets, the envelope glycoprotein,

Viruses are biological entities that are not capable of self-

replicating by themselves, and they require the support of a host

cell transcriptional genetic apparatus for their life cycle. Therefore,

all viruses require that their genetic information (DNA or RNA,

depending upon the Virus typology) reaches the host cell nucleus

for replication. They have developed speciﬁc molecular mecha-

nisms and strategies to achieve that. The blockage of these pro-

cesses prevents virus proliferation and infections. This strategy has

been pursued in virtually all virus pathologies, including Human

Immunodeﬁciency Virus (HIV). Several HIV entry targets have been

gp120, has emerged as a well-validated target [4,5], and its inhi-

bition is critical for bringing additional therapy to HIV infections

[6].

In 2005, we ﬁrst reported identifying a small-molecule inhibitor,

NBD-556, that targets HIV-1 gp120 [7]. However, this inhibitor was

later shown to induce HIV-1 infection in CD4^ꢀCCR5^þcells, thereby

acting as a gp120 agonist [8e10]. Although this was an initial

setback for our endeavor to discover gp120 targeted entry in-

hibitors, we made continuous strides in modifying all three regions

of NBD-556 (Fig. 1). Most notably, we used the scaffold hopping

approach to alter the oxalamide moiety to a diverse scaffold to

convert NBD-556 from being a gp120 agonist to an antagonist. Our

major success came when we replaced the 2,2,6,6-

tetramethylpiperidine scaffold of NBD-556 with a (4-methyl-2-

(piperidin-2-ylmethyl)thiazol-5-yl)methanol scaffold and obtained

NBD-09027. This inhibitor showed partial agonist characteristics.

However, we successfully converted the oxalamide moiety of NBD-

09027 to a pyrrole and kept the other portions in Region I and

Region III (Fig. 1) the same as in NBD-09027. This transformation

Abbreviations: HIV, (Human Immuno Deﬁciency Virus); NDMBA, (N-Nitro-

sodimethylamine); Pd(dba)₂, (Bis(dibenzylideneacetone)palladium); Ro3, (Rule of

3); FBDD, (Fragment-Based Drug discovery).

* Corresponding author.

** Corresponding author.

*** Corresponding author.

E-mail addresses: aaltieri@edasascienti ﬁ c.com (A. Altieri), kurkin@

edasascineti ﬁ c.com (A.V. Kurkin), adebnath@nybc.org (A.K. Debnath).

https://doi.org/10.1016/j.ejmech.2021.113681

I.R. Iusupov, F. Curreli, E.A. Spiridonov et al.

European Journal of Medicinal Chemistry 224 (2021) 113681

Fig. 1. Chronological development of converting a gp120 agonist to a gp120 antagonist [7,9,1 1e16,35] and comparison of the chemical structures of the lead gp120 inhibitors NBD-

556 [7], NBD-09027 [15], NDB-11021 [8], NBD-14136 [14], and NBD-14270 [1 1] and their antiviral activity against pseudovirus HIV-1_HXB-2(IC₅₀), Cytotoxicity (CC₅₀), and selectivity

index (SI).

yielded NBD-11021, which showed a complete reversal of gp120

agonist to gp120 antagonist trait [8], and it was our ﬁrst, second-

generation inhibitor that showed potent antiviral activity (Fig. 1).

Therefore, the scaffold hopping approach achieved our goal of

optimizing NBD-556 from a gp120 agonist to a gp120 antagonist

with potent anti-HIV-1 activity. This report will concentrate on the

scaffold hopping in all three regions, focusing on the region I and III.

One of the recent HIV-1 gp120 entry antagonists, namely NBD-

14270 [1 1], designed in our laboratory, showed excellent antiviral

2.1. Scaffold hopping of the aryl-pyrrole portion via intermediate

acid replacements

In the past, we probed oxalamide-based acids, which led to

compounds behaving as partial gp120 agonists activity (Figs. 1 and

3) [8,16], and one of the most active compounds obtained from the

oxalamide series were NBD-09027 (Figs. 1 and 3) with the para-

chlorophenyl residue on the acid intermediate and the amine

portion constrained in a piperidine ring. The antiviral activity of

these compounds ranged in the low micromolar, but they had

proﬁle and cytotoxicity. It was derived from

a bioisosteric

replacement of the aryl-pyrrole portion in Region I of its analog

NBD-14136 [1 1] (Fig .1) by introducing a pyridine ring in place of the

phenyl and a methyl group on the pyrrole portion. Therefore, we

hypothesize that additional isosteric replacements of this lead

compound may yield inhibitors with improved antiviral potency

and cytotoxicity. The majority of the NBD-based gp120 entry in-

hibitor arrays [2,7,8,1 1e14] were derived from an amine and an acid

portion coupled together via an amide bond, as shown in Fig. 2.

Albeit, the aryl portion of NBD-14136 was mostly explored,

mainly via probing a different kind of aryl moieties [2,8,1 1,13e1 7],

yet other heteroaromatic or different aromatic systems than aryl or

pyridine were not probed. Similarly, the amine portion was not

entirely explored. Herewith in we report a series of new validated

synthetic pathways of the left and right parts (regions I and III) that

enable additional scaffold hopping attempts driven by isosteric

modiﬁcations of NBD-14136 and NBD-14270.

relatively high cytotoxicity (CC₅₀about 30 mM) [8,15]. Since no

single compound derived from the para-chlorophenyl oxalamide

scaffold having, instead of the piperidine a methylenamine moiety

as per the full agonist NBD-14091 was ever tested [7,9,1 1,12,15,16],

we decided to ﬁll the gap and by exploiting promptly available

intermediates from previous works (amines 1a, 2a [14] and acid 1b

(Table 1), thus producing compounds 1 and 2 (as per the procedure

depicted in Scheme 5) in both enantiomeric forms. On top, an

additional elongated analog compound 3 (Table 1), derived from

acid 3c (Scheme 1) with an extra methylene unit and a pCl-mF-

benzyl residue ring, was also made following the general proced-

ures as per Scheme 5. The rational design of compound 3 came

from an attempt to improve compound 4 Sp²character and rigidity

(Fig. 3). Antiviral activity (IC₅₀) of all three pairs of enantiomers

ranged in the 10e20

mM activity with problematic cytotoxicity;

therefore, no further efforts were attempted in this direction

(Table 1).

2. Results and discussion

2.2. Pyrrole “open” form replacements

The design of the alternative scaffolds of acids and amines to be

used in the coupling reaction was done, taking into account several

cycles of molecular modeling [8,15] (data not included), a chemistry

feasibility check, and corresponding reagent availabilities. The

synthetic approach for each new acid and amine is reported in the

chemistry section.

The antiviral activity of the NBD compounds was evaluated in a

single-cycle infection assay by infecting TZM-bl cells with the HIV-

1 NL4-3-HXB2-Luc pseudotyped virus expressing Env of the lab-

adapted HIV-1_HXB-2(CXCR4). The cytotoxicity of the small mole-

cules in TZM-bl cells was measured by the colorimetric XTT

method, as previously described [8,15].

Among the alternative acid replacements, compound 4 (Fig. 3),

an “open” pyrrole form of a previously tested compound NBD-

14091 with a 0.75

m

M IC₅₀[14]^,which required starting a boc-

protected acid 4b (Table 1) was commercially and promptly avail-

able, was considered. Compound 4b undergone coupling and

cleavage reactions against amines 4a(fR) and 4a(fS) [14] [stereo-

chemical descriptor f(R) and f(S) deﬁned in the previous work [13]

as per the general procedures A and B (Section 2.5.), and according

to the pathway depicted in Scheme 5 yielding compounds 4(fS) and

(fR)]. None of these derivatives showed any appreciable antiviral

activity.

Since 4 (fR) and 4 (fS) presented a signiﬁcant loss of activity

2

I.R. Iusupov, F. Curreli, E.A. Spiridonov et al.

European Journal of Medicinal Chemistry 224 (2021) 113681

Fig. 2. Schematic representation of the retrosynthetic approach of the NBD gp120 compound array and how they can be envisioned by a coupling reaction from an Aryl/heteroaryl

pyrrole acid (left part) and a thiazole amine derivative (right part) and the “matrix-like” idea for generating new gp120 orientated inhibitor compounds by using bioisosteric

replacements of the acids and the amine portions. PG ¼ Protecting Group.

compared to NBD-14091 and NBD-14092, we speculated that this

could be attributed to the destruction of the rigidity Sp²pyrrole

saturated system. Therefore, we envisioned compound 3 (Fig. 3), as

mentioned above, and compound 5 (Scheme 2), which could still be

considered as an “open form” of the pyrrole, however being more

rigid due to its extended Sp²urea-like system.

However, compounds 5 and 3, in both enantiomeric forms (fR

and fS) failed to show any signiﬁcant antiviral activity (Table 1 and

Fig. 3), and no other amines were explored against these fragments.

that

4-(cyclohexylmethoxy)phenyl

and

the

4-(cyclo-

hexylmethylamino)phenyl moieties, among other considered,

occupied almost the same distance of the aryl-pyrrole moiety in the

gp120 pocket, at the same time keeping the same crucial key in-

teractions. The two acids were synthesized in good yields as per the

pathway depicted in Scheme 4 to verify if these kinds of re-

placements were fruitful.

These acids, 7d and 8d, were then reacted with the enantiopure

amines 3a(fR) and 3a(fS) [13], accordingly the general procedures A

and B (described in the chemistry section 2.5.) and as per the

depicted in Scheme 5. The resulting products 7(fR), 7(fS), and 8(fR),

8(fS), were poorly active (Table 1). To probe if a slightly different

distance between the phenyl ring and the amide moiety could have

affected the activity, similar analogs of these compounds, namely

9(fR) and 9(fS) (Fig. 3), with an extra methylene unit in between the

phenyl and carbonyl group were also synthesized (general pro-

cedure as per Scheme 5; commercial starting acid as per Table 1),

which, however, also showed no signiﬁcant antiviral activity

(Table 1 - Fig. 3). Therefore these kinds of scaffolds were not pur-

sued any further.

2.3. Phenyl ring replacement with a saturated cyclohexyl system

A glide-base docking study indicates that the phenyl portion of

NBD-14136 seems to occupy a hydrophobic region of the gp120

binding site (Fig. 4a). Therefore, we envisioned its replacement

with a saturated version of it by introducing a cyclohexyl ring to

replace the phenyl ring on the pyrrole acid as per Scheme 3. It is

noteworthy that besides these hydrophobic interactions, the salt-

bridge formation of the primary amine group in these inhibitors

with Asp368 is essential. In addition, the H-bond interaction of the

hydroxyethyl group with Gln429 may also play an important role

(Fig. 4a).

The resulting acid 6c was coupled, as per Scheme 5, against the

enantiopure amines 3a(fS) and 3a(fR), leading respectively to the

target compounds 6(fS) and 6(fR), that unfortunately were poorly

actives (Table 1 and Fig. 3). This leads to the speculation that the Sp²

system of the phenyl ring may play an essential role in the antiviral

2.5. Indole as fused ring system bioisostere of the aryl-pyrrole

portion

As an additional attempt to replace the phenyl-pyrrole part, we

envisioned the possibility of a fused form of the phenyl and pyrrole

rings, such as the indole ring. By molecular modeling such indole

replacements against few previously used amines, we were able to

identify three commercial indole-based acids, namely 10b, 11b, and

12b (Table 1), for which their respective ﬁnal compounds had good

dock scores in the Phe43 cavity of HIV-1 gp120. Therefore, their

synthesis was prompted following the general procedure A and B as

per Scheme 5 and lead to compounds 10, 11, and 12 in both

enantiomeric forms (Fig. 3). Out of them, the enantiomeric pair

12(fR) and 12(fS) reported having a reasonably good antiviral

activity taking part in a possible a

crucial interaction.

p-p interaction or any other

2.4. Aryl-pyrrole replacement with 4-(cyclohexylmethyl-N/O)

phenyl moiety

After considering few chemically viable replacements of the

aryl-pyrrole portion, from a molecular modeling study, it emerged

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I.R. Iusupov, F. Curreli, E.A. Spiridonov et al.

European Journal of Medicinal Chemistry 224 (2021) 113681

Fig. 3. Summary of chemical structures of the NBD-HIV-1 entry inhibitors obtained from aryl-pyrrole replacements of the lead scaffold, their IC₅₀against pseudovirus HIV-1_HXB-2

and CC₅₀proﬁles compared to their similar known analogs, NBD-14091, NBD-14092 [14] NBD-09027 [15], NBD-14108 [12]. The stereochemistry descriptors (fR) and (fS) were

discussed in earlier work [13].

activity (5.1e6.4

14091-2 (0.75e4.3

index with a CC₅₀improvement of nearly two-fold (83e84

versus ~30 M for NBD-14091-2) [13]. Glide-based docking in Fig. 4

m

M), comparable to reference compounds NBD-

phenyl ring is crucial. Thus, rendering the corresponding indole

acid 12b is expected to be a valuable tool for other gp120 inhibitor

scaffold design and explorations.

m

M), however, presenting a better selectivity

mM

m

(b) indicated similar interactions as were observed with NBD-

14136. However, there were some notable differences, too;

namely, the ethyl hydroxy group in the thiazole ring of 12 (fR) forms

2.6. Scaffold hopping via intermediate amine replacements of

unsubstituted thiazole

an H-bond with Trp427. Furthermore, Trp427 forms two

teractions with the phenyl pyrrole part.

p-p in-

While different substituted thiazole-containing compounds

have previously been tested [8,1 1e16], the unsubstituted thiazole

ring was never considered. Therefore we made the corresponding

protected unsubstituted thiazole-based amines 13c(fS) and 13c(fR)

as per the synthetic route in Scheme 6 and eventually coupled them

with acid 13d (Table 1) as per the procedure depicted in Scheme 5.

Interestingly, the resulting enantiomeric products 13(fS) and

The fact that 12 (both fR and fS) had moderate activity, while 10

and 11 were virtually inactive, can be attributed to its Cl in position

6. First, we created the 3D structures of 11 and 12 as described

under section 3.0.2. Then, we used the Ligand Align wizard in

maestro within Schrodinger Suite 2020-4 to overlap NBD-14091

with 11 and 12 as a racemate (Fig. 5). It is evident how the p-Cl

moiety of NBD-14091 occupies a similar place as the 6-Cl-indole

moiety in 12. On the contrary, Cl in 11 points in a different position

in the long but narrow active site. This result points out that the

indole position 6 is more beneﬁcial for substitution than the 5, but

that a lipophilic and small substituent in the para position of the

13(fR) had

(2.4e1.0 M, Fig. 6 and Table 1) to NBD-14091 and NBD-14092

(0.75e1.3 M) [13], rendering this amine a useful reagent tool for

a comparable safety index and antiviral activity

m

further coupling reactions with acids to generate HIV-1 gp120-

targeted inhibitors.

Since the unsubstituted thiazole simpliﬁcation leads to a similar

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European Journal of Medicinal Chemistry 224 (2021) 113681

Table 1

gp120 targeted inhibitors (products) derived from corresponding acids, and amines, and their antiviral activity (IC₅₀) against HIV-1_HXB2, HIV-1_#11578and cell viability values

(CC₅₀).

Amine ID

Amine

Acid ID

Acid Structure

Product ID Product structure

HIV-1_HXB-2IC₅₀

(

m

M) HIV-1_#11578IC₅₀

(

m

M) CC₅₀(mM)

Structure

1a(fR)*, &

1b

1(fR)

>27

>54

1a(fS)*, &

1b

1(fS)

2(fR)

>27

>15

>54

2a(fR)*, &

10.6 0.1

46.6

3

2a(fR)*, &

1b

2(fS)

14.7 1.9

>15

38.3

2

3a(fR) *, &

3c

3(fR)

>15

>77.6

3a(fS) *, &

3c

3(fS)

>15

>77.6

4a(fR) *, &

4b

4(fR)

4(fS)

>23.4

>70

4a(fS) *, &

(continued on next page)

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European Journal of Medicinal Chemistry 224 (2021) 113681

Table 1 (continued)

Amine ID

Amine

Acid ID

Acid Structure

Product ID Product structure

HIV-1_HXB-2IC₅₀

(

m

M) HIV-1_#11578IC₅₀

(

m

M) CC₅₀(mM)

Structure

1a*, &

5c^{^}

5(fR)

>54

1a*, &

5c^{^}

5(fS)

6(fR)

6(fS)

7(fR)

7(fS)

>54

>17

>15

>54

>28

>15

>54

>57

3a(fR)*, &

3a(fS)*, &

3a(fR)*, &

3a(fS)*, &

6c

7d

28.2 0.5

26.5 1.4

3a(fR)*, &

3a(fS)*, &

3a(fR)*, &

8d

9b

8(fR)

8(fS)

9(fR)

>15

>25

>15

>25

56.5

1

56.5 0.5

27.9 0.6

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European Journal of Medicinal Chemistry 224 (2021) 113681

Table 1 (continued)

Amine ID

Amine

Acid ID

Acid Structure

Product ID Product structure

HIV-1_HXB-2IC₅₀

(

m

M) HIV-1_#11578IC₅₀(mM) CC₅₀(mM)

Structure

3a(fS)*, &

9b

9(fS)

>25

47.5 3.8

>60.0

>60

1a(fR)*, &

1a(fS)*, &

1a(fR)*, &

1a(fS *, &

10b

10(fR)

10(fS)

11(fR)

11(fS)

>60

>40

~50

>60

>40

10b

11b

~47

11b ex36

>55

4a(fR)*, &

12b

12(fR)

12(fS)

5.1 0.5

7

0.7

84

1

4aa(fS)*, &

6.4 1.5

8.2 0.3

83.6 3.3

13c(fR)*

13c(fS)*

13d¹⁴

13(fR)

13(fS)

2.4 0.4

3.3 0.3

38.9 1.4

13d¹⁴

1

0.04

0.8 0.3

36.5 1.7

(continued on next page)

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European Journal of Medicinal Chemistry 224 (2021) 113681

Table 1 (continued)

Amine ID

Amine

Structure

Acid ID

Acid Structure

Product ID Product structure

HIV-1_HXB-2IC₅₀

3.3 0.6

(

m

M) HIV-1_#11578IC₅₀

3.8 0.6

(mM) CC₅₀(mM)

14a*

14b¹⁴

14

17.8 0.9

15a*

14b¹⁴

15

16

4.9 0.6

6.6 0.8

9.7 0.5

16

14b¹⁴

13.2 0.6

6.2 0.6

12.8 0.4

17i*

17k¹⁴

17

5

0.2

11.4 1.8

33.7 3.4

18c(fR)

18c(fS)

17k¹⁴

18(fR)

18(fS)

>12.5

12.4 1.9

~40

17k¹⁴

8.5 3.2

(*) All the starred compounds have been reported with the corresponding protecting group/s, which is not present in the ﬁnal product structure since it was cleaved after that

the coupling took place. The actual procedure is well described in the chemistry section for each speciﬁc case.

(&) Intermediate synthesized and reported previously [12].

(^) Not an acid intermediate and ﬁnal product obtained via amine/isocyanate condensation instead. Details are reported in the corresponding chemistry section.

Scheme 1. Depiction of the synthetic approach to acid 3c.

antiviral activity, we decided to explore an even more drastic

simpliﬁcation by completely removing the thiazole ring and testing

compound 14 (Fig. 3), which was obtained by reacting the mono-

boc-protected ethylendinamine 14a (Table 1) against acid 14b

(Table 1 - acid explicitly selected since it was among one of the

best-performing acids from the previous projects [13]) as per the

general procedure A and B (chemistry section 2.5.). Interestingly, 14

the chemical structures, Table 1 for the amine precursors, and the

general coupling procedure A and B as per Section 2.5.) where the

ethylenediamine portion was replaced with moieties containing

piperidine ring. Both compounds had a CC₅₀of about 10

mM, while

the antiviral activity was 4.9 M for 15 and 13.2 M for 16. Clearly,

m

the cytotoxicity and the weak antiviral activity being a limitation

for this kind of variations.

showed antiviral activity at low

1 and Fig. 6), but at the same time demonstrated signiﬁcant cyto-

toxicity (CC₅₀17.8 0.9 M).

The cytotoxicity further deteriorated when we tested a more

rigid isosteric version than 14, like compounds 15 and 16 (Fig. 6 for

m

M range for gp120 inhibition (Tab

Nevertheless, considering that the amine's rigidiﬁcation seemed

to be beneﬁcial for the activity, despite worsening the cell viability,

we envisioned the testing of a “thiazole-containing” scaffolds

having the amine moiety trapped in a piperidine ring such as

compounds 17 and 18 (Fig. 6). Thus, compound 17 was synthesized

m

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European Journal of Medicinal Chemistry 224 (2021) 113681

Scheme 2. Compound 5 synthetic pathway. General procedure B: Pd(dba)2, Ph3P, MeOH.

Fig. 4. Glide-based docking pose of a) NBD-14136 and b) 12 9 fR) bound to a hydrophobic pocket in the HIV-1 envelope glycoprotein gp120. H-bonds and ionic interactions are

indicated by pink arrows, interactions are indicated by green arrows.

p-p

Scheme 3. Synthetic pathway adopted for producing acid 6c.

following the route depicted from Scheme 7.

medicinal chemists for the de-novo design of new scaffolds for

Achieving compound 17 was an exciting result from the merely

synthetic point of view. Its chemistry is novel, and the amine 17h

represents an interesting novel fragment for FBDD, being Ro3

compliant [18] on top of containing privileged drug-like elements

[19]. We have calculated the drug-like properties of all inhibitors

and included them in the Supplementary Materials. Moreover, its

boc-protected version 17i, represent an attractive medicinal

chemistry reagent since it contains at least three synthetic handles

or point of diversity [20e22] that could be expanded judicially.

Therefore, compounds 17h-i represent an interesting tool for any

further synthetic manipulations.

Notably, 17 showed promising antiviral activity (5

mM) with

cytotoxicity (33.7 M) comparable to NBD-14091 and NBD-14092,

m

however presenting the advantage of not being chiral, therefore,

having a more cost-effective synthetic protocol. Thus, this scaffold,

too, along with the indole variation, may represent an alternative

and more economical tool for the chemical space exploration for

the design of HIV-1 gp120-targeted inhibitors.

However, compounds 18(fR) and 18(fS), obtained as per the

synthetic pathway represented in Scheme 8, were not attractive

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European Journal of Medicinal Chemistry 224 (2021) 113681

Scheme 4. Synthetic scheme for acid 7d and 8d.

Scheme 5. Schematic representation of the coupling and cleavage strategies adopted for compounds 1-2,4,6-13. R-COOH as per Table 1.

Fig. 5. Comparison of the chemical structures of NBD-14091, 11, 12 and the 3D-aligned structures of 11 and 12 with NBD-14091.12 was used as the racemate.

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European Journal of Medicinal Chemistry 224 (2021) 113681

Scheme 6. Schematic representation of the synthetic strategy adopted for the alloc protected amines 13c in both enantiomeric forms.

Fig. 6. Chemical structures of ﬁnal compounds 13e18 with their relative antiviral activity against pseudovirus HIV-1_HXB-2(IC₅₀) and cytotoxicity proﬁle (CC₅₀), in comparison with

NBD-556 [7].

due to their low antiviral activity and cell viability proﬁles (Table 1).

As pointed out, the importance of the intermediary compounds

17i-h from the target compound 17, similarly compound 18c, can be

seen in the same way, since it is novel, with low molecular weight

(MW) (241 Da) and containing privileged drug-elements [19].

lab-adapted HIV-1_HXB-2.These ﬁve compounds were further

investigated. At ﬁrst, we tested their speciﬁcity against the control

pseudovirus VSV-G (Table 2) by infecting U87-CD4-CCR5 cells as

previously described [23,24]. We found that 12(fR), 12(fS), 13(fR),

13(fS) were poorly active against VSV-G compared to the activity

we detected against the two HIV-1 clones suggesting that the

inhibitory activity of these compounds is speciﬁc to HIV-1, while

the IC₅₀detected for 17 against this control virus was similar to the

IC₅₀s detected against the HIV-1 clones suggesting that 17 has no

speciﬁcity for HIV-1. These compounds did not induce toxicity in

the U87-CD4-CXCR5 cell line used for this assay.

2.7. Antiviral activity of the NBD compounds against the HIV-

1_{WEAUd15.410.5017}clinical isolate

Antivirals may display different neutralization activity against

laboratory-adapted and clinical isolates HIV-1 clones. Therefore, we

decided to evaluate the activity of the NBD compounds against a

clinical isolate of subtype B, HIV-1_{WEAUd15.410.5017}(NIH # 11578),

which is a dual-tropic (CCR5/CXCR4) clinical isolate. We found that

the IC_50sdetected against this isolate were similar to the IC_50s

detected against the lab-adapted HIV-1_HXB-2(Table 1). Compounds

12(fR), 12(fS), 13(fR), 13(fS), and 17 displayed the best antiviral

activity against the clinical isolate conﬁrming the ﬁndings with the

2.8. Activity of the NBD compounds against HIV-1 Reverse

Transcriptase (RT)

We previously reported that some early generation of NBD

compounds displayed moderate activity against HIV-1 Reverse

Transcriptase (RT) [12,24]. Here, we decided to evaluate the activity

11

I.R. Iusupov, F. Curreli, E.A. Spiridonov et al.

European Journal of Medicinal Chemistry 224 (2021) 113681

Scheme 7. Synthetic route for amine 17i and the ﬁnal product 17. General procedure A: HBTU, DIPEA, DMF. Acid 17k from previous work [1 7].

Scheme 8. Synthetic pathway of compounds 18c and 18. Acid 17k structure as per Table 1.

of the selected ﬁve NBD compounds against that enzyme with a

2.9. NBD compounds are HIV-1 entry-antagonist

colorimetric RT immunoassay (Table 3). We used NBD-556 and

Nevirapine as controls. We found that 12(fR) and 12(fS) alike the

ﬁrst-generation compound NBD-556, had no activity against RT

HIV-1, while 13(fR) and 13(fS) displayed moderate RT inhibition

with an IC₅₀of 31 2.5 and 18.9 4.7 mM, respectively; however, 17

was more effective against RT (IC₅₀of 1.7 0.2). In addition, nevi-

rapine used as positive control showed potent activity against HI-1

The HIV-1 inhibitor NBD-556 [7] was afterward described as an

entry agonist because it promotes CCR5 binding, enhancing HIV-1

entry into CD4-negative cells expressing CCR5 [9,10]. To verify if

the new ﬁve inhibitors behave as an entry antagonist, we infected

CD4-negative Cf2Th-CCR5 cells with the recombinant CD4-

dependant HIV-1_ADAvirus in the presence of escalating concen-

trations of the compounds. NBD-556 (a proven HIV-1 entry agonist)

RT with IC₅₀of <0.5 mM.

12

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European Journal of Medicinal Chemistry 224 (2021) 113681

Table 2

3. Experimental

Antiviral activity of the NBD compounds against control pseudovirus MLV-GP/VSV-

G/Luc (IC₅₀) evaluated in U87-CD4-CCR5 cells and cytotoxicity (CC₅₀).

3.1. Reagents and materials

Compound

U87-CD4-CCR5 cells/VSV-G

Reagents were purchased at the highest commercial quality and

used without further puriﬁcation unless otherwise stated. Starting

materials 1b, 5a-6a, 7a-b, 8a-b, 9b, 10b, 11be12b, 13a-17a and 18a

kindly provided by EDASA scientiﬁc commercial building block

reagents. Reactions were monitored by thin-layer chromatography

(TLC) carried out on Merck TLC Silica gel plates (60F₂₅₄), using a UV

light for visualization and basic aqueous potassium permanganate

or iodine fumes as developing agents. ¹H and ¹³C NMR spectra were

recorded on Bruker Avance 400 instrument with an operating

frequency of 400 and 100 MHz respectively and calibrated using

residual undeuterated chloroform (d_H¼ 7.26 ppm) and CDCl₃

IC₅₀

(

m

M)^a

CC₅₀(m

M)^a

12(fR)

12(fS)

13(fR)

13(fS)

17

15.7

20.5 0.5

12 0.8

16.2 0.5

6.2 0.8

1

>86

45.5 1.5

59.1 0.9

57.7 3.4

a

The reported IC₅₀and CC₅₀values represent the means standard deviations

(n ¼ 3).

Table 3

(

d_C¼ 77.16 ppm), or undeuterated DMSO (d_H¼ 2.50 ppm) and

Activity of NBD compounds against HIV-1 reverse transcriptase

(RT).

DMSO‑d₆

(

d_C¼ 39.51 ppm) as internal references. The following

abbreviations are used to set multiplicities: s ¼ singlet, d ¼ doublet,

t ¼ triplet, q ¼ quartet, quint ¼ quintet, m ¼ multiplet, br. ¼ broad.

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

neat. High-resolution mass spectra (HRMS) were recorded on the

Thermo Scientiﬁc LTQ Orbitrap instrument using nanoelectrospray

ionization (nano-ESI). Low-resolution mass spectra were on Fin-

nigan MAT mass spectrometer using electron ionization (direct

inlet) and an ITD-700 detector with the ionizing electron energy

being 70 eV and the mass range being m/z 35e400. Elemental

analysis was performed on EURO EA CHN Elemental Analyzer. The

melting points (m.p.) were measured in open capillaries and pre-

sented without correction.

Compound

IC₅₀(mM)

NBD-556 (Control)

12(fR)

12(fS)

13(fR)

13(fS)

17

Nevirapine (Control)

>300

>285

245 20

31 2.5

18.9 4.7

1.7 0.2

<0.5

^aThe reported IC₅₀values represent the means standard de-

viations (n ¼ 2).

3.2. GLIDE-based docking

We used the automated docking software GLIDE 8.9 within

€

maestro 12.6 (Schrodinger, Portland, OR) in Schrodinger Suite

2020-4 [25,26] that uses a hierarchical series of ﬁlters in searching

for appropriate ligand conformation in the active site of a target

protein. It uses a funnel-shaped scoring process to sort out the best

conformations and orientations of the ligand (deﬁned as pose)

based on its interactions with the receptor. GLIDE has been used

successfully in drug design [27e31].

We used the crystal structure of clade A/E HIV-1 gp120 core in

complex with NBD-14010 (PDB: 5U6E) [2] that we reported earlier

as the target protein. We used the “Protein Preparation Wizzard”

within maestro in the Schrodinger suite 2020-4 to optimize hy-

drogens, bond orders, charges, and steric clashes using the OPLS3e

force ﬁeld [32]. We used this optimized protein structure to create a

grid ﬁle encompassing the area in the cavity using NBD-14010 as

the ligand.

Fig. 7. Infectivity of Cf2ThꢀCCR5 cells by CD4-dependent HIV-1_ADA. Cf2ThꢀCCR5 cells

were infected with CD4-dependent HIV-1_ADAin the presence of 12(fR) and 13(fR).

NBD-556 and NBD-14270 were used as a control. The Relative virus infectivity des-

ignates the ratio of the amount of infection detected in the presence of the compounds

and the amount of infection detected in the absence of the compounds. Three inde-

pendent experiments were performed in triplicate, and the graph is representative of

one experiment. The toxicity of the compounds against these cells was evaluated to

calculate the CC₅₀values: for NBD-556, the CC₅₀was >60, for NBD-14270 > 47, for

12(fR) > 86 and for 13(fR) 24.7 1.8. All the values represent the mean standard

deviation.

We generated three-dimensional coordinates of the ligands,

their isomeric, ionization, and tautomeric states using the LigPrep

€

(including Ionizer) module within the Schrodinger Suite 2020-4.

Conformational ﬂexibility of the ligands was handled via an

€

exhaustive conformational search. Initially, we used Schrodinger's

and NBD-14270 (a proven HIV-1 entry antagonist [1 1]) were used

as a control. Results are reported in Fig. 7 and expressed as relative

virus infectivity, which designates the ratio of the amount of

infection detected in the presence of the compounds and the

amount of infection detected in the absence of the compounds. As

expected, NBD-556 enhanced the infection of the Cf2Th-CCR5 cells

in a dose-dependent manner, while the control NBD-14270 and the

ﬁve compounds did not enhance HIV-1 infectivity in these cells,

indicating that the HIV-1 entry antagonist property is maintained.

In Fig. 7, data is shown for NBD-556, NBD-14270,12(fR), 13(fR); data

for 12(fS), 13(fS), and 17 is not shown.

proprietary GlideScore scoring function in extra precision (XP)

mode to score the optimized poses.

3.3. Chemistry

3.3.1. General procedure A: for amide coupling

DIPEA (1 equiv.) was added to an appropriate acid (1 equiv.)

followed by dissolving in DMF (10 mL per 1 g of acid) and then

HBTU (1 equiv.). The resulting solution was stirred for 1e3 min and

added to a solution of appropriate amine (1 equiv.) in DMF (10 mL

per 1 g of amine) in several portions. The reaction mixture was

stirred overnight; DMF was evaporated, and the residue was

13

I.R. Iusupov, F. Curreli, E.A. Spiridonov et al.

European Journal of Medicinal Chemistry 224 (2021) 113681

dissolved in CH₂Cl₂(50 mL per 1 g of crude product) and succes-

sively washed with 5% aqueous NaOH and 10% tartaric acid solu-

tions (25 mL per 1 g of crude product). The organic layer was dried

over anhydrous Na₂SO₄, ﬁltered, evaporated, and dry loaded on

silica. The crude product was puriﬁed by ﬂash column chroma-

tography on silica gel (eluent: hexane-EtOAc, 3:1 / 1:1 / 0:1).

Products were used in the next step without analysis.

found 383.0939.

3.3.5. Ethyl 2-[(4-chloro-3-ﬂuoro-phenyl)methylamino]-2-oxo-

acetate (3b)

Amine salt (16.68 g. 85.1 mmol) was suspended in CH₂Cl₂

(170 mL), and Et₃N (30 mL, 215 mmol, 2.5 equiv) was added. Ethyl

chlorooxoacetate (11.4 mL, 102 mmol, 1.2 equiv) was added drop-

wise. The reaction mixture was stirred for 1 h and diluted with 5%

aqueous HCl (200 mL). The organic layer was separated, dried over

Na₂SO₄, ﬁltered, and evaporated. The residue was used without

further puriﬁcation. If necessary, the product can be puriﬁed by

column chromatography (eluent: 3:1, 1:1 hexanes\EtOAc).

M ¼ 10.17 g. Yield ¼ 46%.

3.3.2. General procedure B: for allyl- and alloc- deprotection

To a solution containing protected compound (1 equiv.) and

N,N-dimethyl barbituric acid (NDMBA, 3 equiv.) in MeOH (0.1 M

solution), PPh₃(10 mol %) was added under a nitrogen atmosphere

followed by Pd(dba)₂(5 mol %). The mixture was stirred for 1 day

under reﬂux. After cooling, 50 mL of CH₂Cl₂was added, and the

organic phase was shaken with 10% aqueous K₂CO₃(50 mL) to

remove the unreacted NDMBA. The organic layer was separated,

and the aqueous layer was extracted with CH₂Cl₂-EtOH (~4:1,

(4e5) ꢁ 50 mL). Combined organic layers were dried over anhy-

drous Na₂SO₄, ﬁltered, and concentrated. Puriﬁcation by ﬂash

chromatography (twice) (eluent 1: CHCl₃-MeOH (saturated with

NH₃~7 M), 10:1 and 5:1, eluent 2: CH₂Cl₂-MeOH, 4:1, 2:1, 1:1)

afforded amine as a slightly yellow or white solid.

¹H NMR: (CDCl₃, 400 MHz)

d

¼ 1.41 (t, J ¼ 7.2 Hz, 3 H), 4.37 (q,

J ¼ 7.0 Hz, 2 H), 4.51 (d, J ¼ 6.4 Hz, 2 H), 7.05 (d, J ¼ 8.3 Hz, 1 H), 7.12

(dd, J ¼ 9.7, 1.4 Hz, 1 H), 7.38 (t, J ¼ 7.8 Hz, 1 H), 7.55 (br. s, 1 H).

¹³C NMR: (CDCl₃, 100 MHz)

d

¼ 14.0, 42.9, 63.5, 116.2 (d,

J ¼ 21.7 Hz), 120.4 (d, J ¼ 17.7 Hz), 124.3 (d, J ¼ 3.2 Hz), 130.9, 137.9

(d, J ¼ 6.4 Hz), 156.8, 158.1 (d, J ¼ 249.8 Hz), 160.5.

3.3.6. 2-[(4-Chloro-3-ﬂuoro-phenyl)methylamino]-2-oxo-acetic

acid (3c)

Appropriate ethyl ester 3b (10.17 g, 39.1 mmol, 1 equiv.) was

added to a solution of NaOH (3.14 g, 78.5 mmol, 2 equiv.) in EtOH-

H₂O mixture (1:1, 80 mL). The resulting reaction mixture was

reﬂuxed for 5e6 h (TLC-control) and cooled to room temperature. A

concentrated aqueous HCl solution (~12 M, 6.5 mL, 78.5 mmol, 2

equiv.). The reaction mixture was diluted with water (100 mL), the

formed precipitate was ﬁltered and washed with water (2 ꢁ 50 mL).

The pure product was dried under reduced pressure. M ¼ 4.21 g.

Yield ¼ 46%.

3.3.3. N¹-(2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)

ethyl)-N²-(4-chlorophenyl)oxalamide (1)

Compounds 1(fR), NBD-14081, and 1(fS), NBD-14082 were ob-

tained following the general procedure A and B from amines

1a(fR), 1a(fS), and acid 1b. Both compounds were puriﬁed using

column chromatography on silica gel. Eluent CHCl₃-MeOH satu-

rated with NH₃(10:1 and 5:1).

1(fR), NBD-14081: M ¼ 156 mg. Yield ¼ 24% (over two steps).

rt ¼ 1.164 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 369 Da.

1(fS), NBD-14082: M ¼ 291 mg. Yield ¼ 39% (over two steps).

rt ¼ 1.174 min. Purity ¼ 99%. LC-MS: m/z [MþH]^þ¼ 369 Da.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 4.30 (d, J ¼ 6.4 Hz, 2 H), 7.12

(d, J ¼ 8.2 Hz, 1 H), 7.28 (dd, J ¼ 10.4, 1.3 Hz, 1 H), 7.51 (t, J ¼ 8.0 Hz,

1 H), 8.61 (br. s., 1 H), 9.32 (t, J ¼ 6.1 Hz, 1 H).

¹³C NMR (DMSO‑d₆, 100 MHz):

117.8 (d, J ¼ 17.7 Hz), 124.6 (d, J ¼ 3.2 Hz), 130.5, 140.8 (d, J ¼ 6.4 Hz),

157.1 (d, J ¼ 246.6 Hz), 159.8, 162.3.

d

¼ 41.6, 115.7 (d, J ¼ 20.9 Hz),

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 1.75 (br. s., 2 H), 2.25 (s, 3 H),

3.06 (d, J ¼ 6.36 Hz, 2 H), 4.54 (s, 2 H), 5.01 (t, J ¼ 5.72 Hz, 1 H), 5.39

(br. s., 1 H), 7.42 (d, J ¼ 8.90 Hz, 2 H), 7.87 (d, J ¼ 8.90 Hz, 2 H), 9.41

(br. s., 1 H), 10.84 (br. s., 1 H).

3.3.7. N'-[2-amino-1-[5-(hydroxymethyl)thiazol-2-yl]ethyl]-N-[(4-

chloro-3-ﬂuoro-phenyl)methyl]oxamide (3)

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 14.9, 45.1, 54.9, 55.1, 122.1

Compounds 3(fR), NBD-14314 and 3(fS), NBD-14313 were ob-

tained following the general procedure A and B from amines 1a(fR)

and 1a(fS), and acid 3c. Compounds were puriﬁed using column

chromatography on silica gel. Eluent CHCl₃-MeOH saturated with

NH₃(10:1 and 5:1).

(2C), 128.4, 128.7 (2C), 132.9, 136.6, 146.9, 158.4, 160.2, 167.2.

HRMS (ESI): m/z calcd for C₁₅H₁₈ClN₄O₃S [MþH]^þ369.0783,

found 369.0793.

3.3.4. N¹-(3-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)

propyl)-N²-(4-chlorophenyl)oxalamide (2)

Compounds 2(fR), NBD-14079 and 2(fS), NBD-14080 were ob-

tained following the general procedure A and B from amines

2a(fR), 2a(fS), and acid 1b. Compounds were puriﬁed using column

chromatography on silica gel. Eluent CHCl₃-MeOH saturated with

NH₃(10:1 and 5:1).

3(fR), NBD-14314: M ¼ 200 mg. Yield ¼ 21% (over two steps).

rt ¼ 1.214 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 387 Da.

3(fS), NBD-14313: M ¼ 195 mg. Yield ¼ 19% (over two steps).

rt ¼ 1.198 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 387 Da.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 1.74 (br. s., 2 H), 3.00e3.08

(m, 2 H), 4.28e4.41 (m, 2 H), 4.61 (d, J ¼ 5.5 Hz, 2 H), 4.98e5.04 (m,

1 H), 5.49 (t, J ¼ 5.7 Hz, 1 H), 7.15 (dd, J ¼ 8.2, 1.3 Hz, 1 H), 7.32 (dd,

J ¼ 10.3, 1.8 Hz, 1 H), 7.51e7.58 (m, 2 H), 9.28 (br. s., 1 H), 9.48 (t,

J ¼ 6.5 Hz, 1 H).

2(fR), NBD-14079: M ¼ 323 mg. Yield ¼ 22% (over two steps).

rt ¼ 1.138 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 383 Da.

2(fS), NBD-14080: M ¼ 177 mg. Yield ¼ 23% (over two steps).

rt ¼ 1.071 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 383 Da.

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 41.6, 45.1, 54.7, 55.7, 115.8 (d,

J ¼ 20.9 Hz), 117.9 (d, J ¼ 17.7 Hz), 124.6 (d, J ¼ 3.2 Hz), 130.5, 139.0,

140.3,140.5 (d, J ¼ 6.4 Hz),157.0 (d, J ¼ 246.6 Hz),160.0,160.2,170.0.

HRMS (ESI) calcd for C₁₅H₁₇ClF₆N₄O₃S [M þH]^þ387.0688, found

387.0690.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 2.05 (q, J ¼ 6.4 Hz, 2 H), 2.26

(s, 3 H), 2.53e2.63 (m, 1 H), 2.65e2.73 (m, 1 H), 4.55 (s, 2 H), 5.25 (t,

J ¼ 6.8 Hz, 1 H), 5.42 (br. s., 1 H), 7.43 (d, J ¼ 8.9 Hz, 2 H), 7.87 (d,

J ¼ 8.9 Hz, 2 H). (Four exchangeable protons are missing).

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 14.8, 36.6, 38.1, 50.3, 55.0,

3.3.8. N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-2-((4-

chlorobenzyl)amino)acetamide dihydrochloride (4)

122.0 (2C), 128.3, 128.6 (2C), 132.7, 136.6, 146.8, 158.4, 159.9, 168.8.

HRMS (ESI): m/z calcd for C₁₆H₂₀ClN₄O₃S [MþH]þ 383.0939,

Compounds 4(fR), NBD-14155 and 4(fS), NBD-14154 were

14

I.R. Iusupov, F. Curreli, E.A. Spiridonov et al.

European Journal of Medicinal Chemistry 224 (2021) 113681

obtained following the general procedure A and B and followed by

Boc-deprotection (see paragraph below) from amines 3a(fR), 3a(fS),

and acid 4b. The resulting ﬁnal compounds were puriﬁed after

allyl- and alloc-deprotection (gen. proc. B) using column chroma-

tography on silica gel. Eluent CHCl₃-MeOH saturated with NH₃

(10:1).

Boc-deprotection: To Boc-protected compound was added 50 mL

of 1 M HCl in methanol. The solution was stirred for 2 h at room

temperature, and then the solvent was evaporated. The precipitate

was triturated in Et₂O (50 mL) and ﬁltered. The product was

washed with Et₂O (2 ꢁ 50 mL) and CH₂Cl₂(2 ꢁ 50 mL) and dried on

a rotary evaporator under reduced pressure.

The crude product was puriﬁed by ﬂash column chromatography

on silica gel (eluent: hexane-EtOAc, 3:1 / 1.1 / 0:1). The product

was used without analysis.

Allyl- and alloc-deprotection was carried out according to the

general procedure B.

5(fR), (NBD-14087): M ¼ 117 mg. Yield ¼ 15% (over two steps).

rt ¼ 1.134 min. Purity ¼ 95%. LC-MS: m/z [MþH]^þ¼ 369 Da.

5(fS), (NBD-14087): M ¼ 245 mg. Yield ¼ 29% (over two steps).

rt ¼ 1.076 min. Purity ¼ 92%. LC-MS: m/z [MþH]^þ¼ 369 Da.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 2.27 (s, 3 H), 2.95e3.02 (m,

1 H), 3.06e3.13 (m, 1 H), 3.35 (br. s, 3H), 4.55 (s, 2 H), 4.98e5.07 (m,

1 H), 5.41 (br. s., 1 H), 7.59 (d, J ¼ 8.58 Hz, 2 H), 8.00 (d, J ¼ 8.58 Hz,

2 H), 9.40 (d, J ¼ 7.95 Hz, 1 H). (Three exchangeable proton signals

are missing).

4(fR), NBD-14155: M ¼ 481 mg. Yield ¼ 41% (over two steps).

rt ¼ 0.663 min. Purity ¼ 97%. LC-MS: m/z [MþH]^þ¼ 355 Da.

4(fS), NBD-14154: M ¼ 552 mg. Yield ¼ 55% (over two steps).

rt ¼ 0.793 min. Purity ¼ 99%. LC-MS: m/z [MþH]^þ¼ 355 Da.

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 14.9, 45.5, 53.8, 55.1, 128.7

(2 C), 130.2 (2 C), 131.3, 132.9, 137.8, 147.1, 153.3, 167.3, 168.1.

HRMS (ESI): m/z calcd for C₁₅H₁₈ClN₄O₃S [MþH]^þ369.0783,

found 369.0782.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 3.19e3.30 (m, 1 H),

3.38e3.51 (m, 1 H), 3.70e3.88 (m, 2 H), 4.13e4.25 (m, 2 H), 4.63 (s,

2 H), 5.42e5.50 (m, 1 H), 7.49 (d, J ¼ 8.31 Hz, 2 H), 7.55e7.63 (m,

3 H), 8.57 (br. s., 3 H), 9.69 (d, J ¼ 8.19 Hz, 1 H), 9.81 (br. s., 2 H). (One

exchangeable proton is missing).

3.3.12. 1-(5-Cyclohexyl-1H-pyrrol-2-yl)-2,2,2-triﬂuoroethanone

(6b)

Pyrrole 6a (8.95 g, 60.0 mmol, 1 equiv.) was dissolved in CH₂Cl₂

(120 mL), and pyridine (5.83 mL, 72.1 mmol, 1.2 equiv.) was added,

followed by dropwise addition of TFAA (10.02 mL, 72.1 mmol, 1.2

equiv.). After completion of the addition, the mixture was stirred

for 2e3 h at room temperature, and the solvent was evaporated.

The crude product was puriﬁed by ﬂash chromatography on silica

gel (eluent: hexane-EtOAc, 30:1 / 20:1). M ¼ 9.58 g. Yield ¼ 65%.

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 41.4, 47.0, 49.0, 49.1, 55.7,

128.7 (2 C), 130.6, 132.4 (2 C), 133.9, 139.1, 142.0, 165.7, 167.2.

HRMS (ESI): m/z calcd for C₁₅H₁₉ClN₄O₂S [MþH]^þ355.0990,

found 355.0993.

3.3.9. 4-Chlorobenzamide (5b)

¹H NMR: (CDCl₃, 400 MHz)

d

¼ 1.20e1.33 (m, 1H), 1.33e1.54 (m,

4-Chlorobenzoic acid (14.8 g, 94.9 mmol, 1 equiv.) was dissolved

in CHCl₃(95 mL), Et₃N (15.54 mL, 104.6 mmol, 1.1 equiv.) was added

in a single portion followed by dropwise addition of ethyl chlor-

oformate (10.0 mL, 104.6 mmol, 1.1 equiv.). After the addition, the

mixture was cooled to 0 ^ꢂC, and aqueous ammonia (50 mL) was

slowly added. The mixture was stirred for 1 h at RT, and layers were

separated. The aqueous phase was extracted with CH₂Cl₂

(2 ꢁ 50 mL). Combined organic layers were washed with water

(50 mL), brine (50 mL), dried over anhydrous Na₂SO₄, ﬁltered, and

evaporated. The product was used in the next step without further

puriﬁcation. M ¼ 7.90 g. Yield ¼ 54%.

4H), 1.69e1.79 (m, 1H), 1.80e1.89 (m, 2H), 1.96e2.09 (m, 2H), 2.75

(tt, J ¼ 11.3, 3.2 Hz, 1H), 6.18 (dd, J ¼ 4.0, 2.6 Hz, 1H), 7.12e7.25 (m,

1H), 10.49 (br. s, 1H).

¹³C NMR: (CDCl₃, 100 MHz)

d

¼ 25.8, 26.1, 32.5, 37.5, 109.8, 117.5

(q, J ¼ 288.3 Hz), 123.7 (q, J ¼ 3.4 Hz), 124.9, 152.6, 168.9, 169.1 (q,

J ¼ 35.9 Hz).

3.3.13. 5-Cyclohexyl-1H-pyrrole-2-carboxylic acid (6c)

Appropriate triﬂuoroethanone 6b (2.58 g, 10.5 mmol, 1 equiv.)

was added to a solution of NaOH (2.1 g, 52.6 mmol, 5 equiv.) in

EtOH-H₂O mixture (1:1, 53 mL). The resulting reaction mixture was

reﬂuxed for 20 h and cooled to room temperature. A concentrated

aqueous HCl solution (~12 M, 5 equiv.) was added dropwise, and

water (50 mL) was added. The resulting mixture was extracted with

CH₂Cl₂(3 ꢁ 100 mL). Combined organic phases were dried over

anhydrous Na₂SO₄, ﬁltered, and evaporated. The crude product was

puriﬁed by ﬂash chromatography on silica gel (eluent: hexane-

EtOAc, 1:1 / 0:1) to afford 5. M ¼ 1.69 g. Yield ¼ 83%.

¹H NMR: (DMSO‑d₆, 400 MHz)

J ¼ 8.3 Hz, 2 H), 7.89 (d, J ¼ 8.3 Hz, 2 H), 8.06 (br. s, 1 H).

d

¼ 7.47 (br. s, 1 H), 7.52 (d,

3.3.10. 4-Chlorobenzoyl isocyanate (5c)

Amide 24 (7.9 g, 51.0 mmol, 1 equiv.) was dissolved in 1,2-

dichloroethane (51 mL), and oxalyl chloride (22 mL, 260 mmol,

5.0 equiv) was added in a single portion. The mixture was heated at

reﬂux for 1 day and cooled to room temperature. The resulting

solution was evaporated to dryness. The product was used in the

next step without further puriﬁcation. M ¼ 9.23 g. Yield ¼ 100%.

¹H NMR (CDCl₃, 400 MHz):

d

¼ 1.22e1.48 (m, 5 H),1.68e1.87 (m,

3 H), 1.94e2.05 (m, 2 H), 2.55e2.70 (m, 1 H), 5.99e6.07 (m, 1 H),

6.96e7.03 (m, 1 H), 9.14 (br. s., 1 H), 10.92 (br. s., 1 H).

¹H NMR (CDCl₃, 400 MHz):

d

¼ 7.47 (d, 2 H), 8.01 (d, J ¼ 7.9 Hz,

¹³C NMR (CDCl₃, 400 MHz):

d

¼ 26.0, 26.2 (2 C), 32.9 (2 C), 37.1,

2 H).

107.1, 118.3, 119.9, 145.6, 166.1.

¹³C NMR (DMSO‑d₆,100 MHz):

d

¼ 129.3 (2 C),129.9,131.6,131.9

(2 C), 144.6, 164.3.

3.3.14. N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-5-

cyclohexyl-1H-pyrrole-2-carboxamide (6)

3.3.11. N-((2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)

ethyl)carbamoyl)-4-chlorobenzamide (5)

Compounds 6(fR), NBD-14146 and 6(fS), NBD-14147 were

respectively obtained following the general procedure A and B

from amines 3a(fR), 3a(fS), and acid 6c. Compounds were puriﬁed

using column chromatography on silica gel. Eluent CHCl₃-MeOH

saturated with NH₃(10:1 and 5:1).

To a solution of amine 1a (0.7 g, 2.25 mmol, 1 equiv.) in MeCN

(10 mL), 4-chlorobenzoyl isocyanate 5c (0.45 g, 2.48 mmol, 1.1

equiv.) was added, followed by a catalytic amount of DMAP. The

mixture was stirred for 30 min at room temperature and then

reﬂuxed for 4e5 h (TLC-control). After that, the solvent was evap-

orated, and the residue was dissolved in CH₂Cl₂(50 mL) and

washed with 10% aqueous tartaric acid (25 mL). The organic layer

was separated, dried over anhydrous Na₂SO₄, ﬁltered, evaporated.

6(fR), NBD-14146: M ¼ 87 mg. Yield ¼ 8% (over two steps).

rt ¼ 1.222 min. Purity ¼ 95%. LC-MS: m/z [MþH]^þ¼ 349 Da.

6(fS), NBD-14147: M ¼ 275 mg. Yield ¼ 16% (over two steps).

rt ¼ 1.055 min. Purity ¼ 96%. LC-MS: m/z [MþH]^þ¼ 349 Da.

15

I.R. Iusupov, F. Curreli, E.A. Spiridonov et al.

European Journal of Medicinal Chemistry 224 (2021) 113681

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 1.11e1.42 (m, 5 H), 1.45e1.92

37.0, 73.0, 114.2 (2 C), 122.8, 131.4 (2 C), 162.5, 167.1.

(m, 7 H), 2.53e2.61 (m, 1 H), 2.97 (dd, J ¼ 13.14, 7.89 Hz, 1 H), 3.10

(dd, J ¼ 13.20, 5.26 Hz,1 H), 4.60 (s, 2 H), 5.10e5.19 (m,1 H), 5.47 (br.

s., 1 H), 5.81e5.85 (m, 1 H), 6.77e6.82 (m, 1 H), 7.53 (s, 1 H), 8.29 (d,

J ¼ 7.95 Hz, 1 H), 11.17 (br. s., 1 H).

3.3.18. 4-((cyclohexylmethyl)amino)benzoic acid (8d)

Appropriate methyl ester 8c (5.76 g, 23.3 mmol, 1 equiv.) was

added to a solution of NaOH (1.86 g, 46.6 mmol, 2 equiv.) in EtOH-

H₂O mixture (1:1, 35 mL). The resulting reaction mixture was

reﬂuxed for 5e6 h (TLC-control) and cooled to room temperature. A

concentrated aqueous HCl solution (~12 M, 3.88 mL, 2 equiv.) was

added dropwise, and water (50 mL) was added. The precipitate that

formed was ﬁltered, washed with water (2 ꢁ 50 mL). The pure

product was dried under reduced pressure. M ¼ 4.71 g. Yield ¼ 87%.

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 25.6, 25.9 (2 C), 32.7, 32.7,

36.3, 45.7, 54.3, 55.8, 104.1, 111.3, 124.1, 139.0, 139.9, 142.4, 160.8,

172.5.

HRMS (ESI): m/z calcd for C₂₁H₃₀N₃O₃S [MþH]^þ349.1693, found

349.1695.

3.3.15. Methyl 4-(cyclohexylmethoxy)benzoate (7c)

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 0.85e0.98 (m, 2 H),

Methyl 4-hydroxybenzoate 7a (19.78 g, 130 mmol, 1 equiv.) and

cyclohexylmethyl methanesulfonate 7b (25 g, 130 mmol, 1 equiv.)

were dissolved in DMF (260 mL). Solid K₂CO₃(35.89 g, 260 mmol, 2

equiv.) was added to a solution followed by NaI*2H₂O (2.42 g,

13.0 mmol, 0.1 equiv.) and TBAB (4.19 g, 13.0 mmol, 0.1 equiv.)

addition. The reaction mixture was stirred for 12e15 h at 90e100 ^ꢂC

on an oil bath. After cooling to room temperature, the mixture was

poured into water (550 mL) and extracted with EtOAc (3 ꢁ 200 mL).

Combined organic phases were washed with water (150 mL) and

brine (2 ꢁ 100 mL), dried over anhydrous Na₂SO₄, ﬁltered, and

evaporated. The crude product was puriﬁed by ﬂash chromatog-

1.08e1.24 (m, 3 H), 1.44e1.82 (m, 6 H), 2.89 (t, J ¼ 6.05 Hz, 2 H), 6.44

(t, J ¼ 5.44 Hz, 1 H), 6.54 (d, J ¼ 8.68 Hz, 2 H), 7.64 (d, J ¼ 8.68 Hz,

2 H), 11.88 (br. s., 1 H).

¹³C NMR (DMSO‑d₆, 100 MHz):

36.9, 49.0, 110.7 (2 C), 116.4, 131.3 (2 C), 153.0, 167.7.

d

¼ 25.6 (2 C), 26.2, 30.7 (2 C),

3.3.19. N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-4-

(cyclohexylmethoxy)benzamide (7)

Compounds 7(fR), NBD-14216 and 7(fS), NBD-14217) were ob-

tained following the general procedure A and B from amines

3a(fR), 3a(fS), and acid 7d. Compounds were puriﬁed using column

chromatography on silica gel. Eluent CHCl₃-MeOH saturated with

NH₃(10:1 and 5:1).

raphy on silica gel (eluent: hexane-EtOAc, 20:1

/ 10:1).

M ¼ 24.57 g. Yield ¼ 76%.

¹H NMR (CDCl₃, 400 MHz):

d

¼ 0.99e1.13 (m, 2 H), 1.17e1.38 (m,

3 H), 1.65e1.93 (m, 6 H), 3.80 (d, J ¼ 6.24 Hz, 2 H), 3.88 (s, 3 H), 6.90

7(fR), NBD-14216: M ¼ 596 mg. Yield ¼ 53% (over two steps)

rt ¼ 1.357 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 390 Da.

7(fS), NBD-14217): M ¼ 610 mg. Yield ¼ 47% (over two steps).

rt ¼ 1.363 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 390 Da.

(d, J ¼ 8.56 Hz, 2 H), 7.98 (d, J ¼ 8.56 Hz, 2 H).

¹³C NMR (CDCl₃, 100 MHz):

d

¼ 25.8 (2 C), 26.5, 29.9 (2 C), 37.7,

51.8, 73.6, 114.1 (2 C), 122.3, 131.6 (2 C), 163.2, 167.0.

3.3.16. Methyl 4-((cyclohexylmethyl)amino)benzoate (8c)

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 0.97e1.10 (m, 2 H), 1.13e1.32

To a solution of methyl 4-aminobenzoate (34.7 g, 230 mmol, 1

equiv.) in CH₂Cl₂(460 mL) on an ice bath, cyclohexanecarbaldehyde

(25.75 g, 230 mmol, 1 equiv.) was added, followed by dropwise

addition of AcOH (1.31 mL, 23.0 mmol, 0.1 equiv.). The mixture was

stirred for 10e15 min, and then NaBH(OAc)₃(121.6 g, 574 mmol, 2.5

equiv.) was added portion-wise at the same temperature. The

resulting mixture was stirred for 1 h at 0e5 ^ꢂC and 10e12 h at room

temperature; after that, it was poured (carefully! CO₂evolution) into

10% aqueous K₂CO₃(1000 mL). The organic layer was separated,

and water was extracted with CH₂Cl₂(3 ꢁ 150 mL). Combined

organic phases were washed with brine (200 mL), dried over

anhydrous Na₂SO₄, ﬁltered, and evaporated. The crude product was

puriﬁed by ﬂash column chromatography on silica gel (eluent:

hexane-EtOAc, 20:1 / 10:1). M ¼ 45.29 g. Yield ¼ 80%.

(m, 3 H), 1.49e1.92 (m, 8 H), 3.00 (dd, J ¼ 13.08, 8.07 Hz, 1 H), 3.11

(dd, J ¼ 13.20, 5.01 Hz, 1 H), 3.85 (d, J ¼ 5.99 Hz, 2 H), 4.60 (s, 2 H),

5.08e5.24 (m, 1 H), 5.47 (br. s., 1 H), 7.01 (d, J ¼ 8.56 Hz, 2 H), 7.54 (s,

1 H), 7.89 (d, J ¼ 8.68 Hz, 2 H), 8.79 (d, J ¼ 7.21 Hz, 1 H).

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 25.3 (2 C), 26.0, 29.2 (2 C),

37.0, 45.6, 55.3, 55.8, 72.9, 113.9 (2 C), 126.1, 129.4 (2 C), 139.0, 139.9,

161.4, 166.2, 172.2.

HRMS (ESI): m/z calcd for C₂₀H₂₈N₃O₃S [MþH]^þ390.1846, found

390.1850.

3.3.20. N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-4-

((cyclohexylmethyl)amino)benzamide (8)

Compounds 8(fR), NBD-14218 and 8(fS), NBD-14219 were ob-

tained following the general procedure A and B from amines

3a(fR), 3a(fS), and acid 8d. Compounds were puriﬁed using column

chromatography on silica gel. Eluent CHCl₃-MeOH saturated with

NH₃(10:1 and 5:1).

¹H NMR (CDCl₃, 400 MHz):

d

¼ 0.91e1.03 (m, 2 H), 1.12e1.31 (m,

3 H), 1.51e1.84 (m, 6 H), 2.99 (d, J ¼ 6.72 Hz, 2 H), 3.84 (s, 3 H), 4.28

(br. s., 1 H), 6.50e6.55 (m, 2 H), 7.82e7.87 (m, 2 H).

¹³C NMR (CDCl₃, 100 MHz):

d

¼ 26.0 (2 C), 26.5, 31.2 (2 C), 37.6,

49.9, 51.5, 111.3 (2 C), 117.7, 131.6 (2 C), 152.4, 167.5.

8(fR), NBD-14218: M ¼ 785 mg. Yield ¼ 44% (over two steps).

rt ¼ 1.366 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 389 Da.

8(fS), NBD-14219: M ¼ 684 mg. Yield ¼ 38% (over two steps).

rt ¼ 1.338 min. Purity ¼ 98%. LC-MS: m/z [MþH]^þ¼ 389 Da.

3.3.17. 4-(cyclohexylmethoxy)benzoic acid (7d)

Appropriate methyl ester 7c (24.57 g, 98.9 mmol, 1 equiv.) was

added to a solution of NaOH (7.92 g, 198 mmol, 2 equiv.) in EtOH-

H₂O mixture (1:1, 200 mL). The resulting reaction mixture was

reﬂuxed for 7e8 h (TLC-control) and cooled to room temperature. A

concentrated aqueous HCl solution (~12 M, 16.5 mL, 2 equiv.) was

added dropwise, and water (200 mL) was added. The precipitate

that formed was ﬁltered, washed with water (2 ꢁ 50 mL). The crude

product was recrystallized from EtOH. M ¼ 21.39 g. Yield ¼ 92%.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 0.87e1.01 (m, 2 H), 1.10e1.26

(m, 3 H), 1.39e1.86 (m, 8 H), 2.91 (t, J ¼ 6.19 Hz, 2 H), 3.00 (dd,

J ¼ 13.14, 7.83 Hz, 1 H), 3.10 (dd, J ¼ 13.14, 5.05 Hz, 1 H), 4.60 (s, 2 H),

5.16 (td, J ¼ 7.64, 5.31 Hz, 1 H), 5.47 (br. s., 1 H), 6.25 (t, J ¼ 5.62 Hz,

1 H), 6.58 (d, J ¼ 8.72 Hz, 2 H), 7.53 (s, 1 H), 7.70 (d, J ¼ 8.72 Hz, 2 H),

8.46 (d, J ¼ 7.71 Hz, 1 H).

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 0.90e1.06 (m, 2 H), 1.09e1.31

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 25.6 (2 C), 26.2, 30.7 (2 C),

(m, 3 H), 1.55e1.83 (m, 6 H), 3.78 (d, J ¼ 6.11 Hz, 2 H), 6.96 (d,

J ¼ 8.80 Hz, 2 H), 7.88 (d, J ¼ 8.68 Hz, 2 H), 12.60 (br. s., 1 H).

36.9, 45.7, 49.0, 55.0, 55.8, 110.5 (2 C), 120.1, 129.1 (2 C), 139.0, 139.8,

151.9, 166.7, 172.9.

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 25.3 (2 C), 26.1, 29.2 (2 C),

HRMS (ESI): m/z calcd for C₂₀H₂₉N₄O₂S [MþH]^þ389.2006,

16

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European Journal of Medicinal Chemistry 224 (2021) 113681

found 389.2009.

4.52 (s, 2 H), 5.16e5.25 (m, 1 H), 5.37 (br. s., 1 H), 7.19 (dd, J ¼ 8.8,

2.0 Hz, 1 H), 7.28 (s, 1 H), 7.43 (d, J ¼ 8.8 Hz, 1 H), 7.73 (d, J ¼ 1.8 Hz,

1 H), 9.03 (s, 1 H), 11.84 (br. s., 1 H).

3.3.21. N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-2-(4-

(cyclohexylmethoxy)phenyl)acetamide (9)

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 14.9, 45.2, 54.2, 55.0, 103.1,

Compounds 9(fR), NBD-14157 and 9(fS), NBD-14156 were ob-

tained following the general procedure A and B from amines

3a(fR), 3a(fS), and acid 9b. Compounds were puriﬁed using column

chromatography on silica gel. Eluent CHCl₃-MeOH saturated with

NH₃(10:1 and 5:1).

113.9, 120.7, 123.7, 124.3, 128.1, 132.6, 132.7, 135.0, 147.0, 161.0, 168.6.

HRMS (ESI): m/z calcd for C₁₆H₁₈ClN₄O₂S [MþH]^þ365.0834,

found 365.0830.

3.3.24. N-(2-amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-6-

chloro-1H-indole-2-carboxamide (12)

9(fR), NBD-14157: M ¼ 35 mg. Yield ¼ 8% (over two steps).

rt ¼ 1.443 min. Purity ¼ 93%. LC-MS: m/z [MþH]^þ¼ 404 Da.

9(fS), NBD-14156: M ¼ 448 mg. Yield ¼ 50% (over two steps).

rt ¼ 1.402 min. Purity ¼ 94%. LC-MS: m/z [MþH]^þ¼ 404 Da.

Compounds 12(fR), NBD-14294 and 12(fS), NBD-14293 were

obtained following the general procedure A and B from amine

12a(fR), 12a(fS), and acid 12b. Compounds were puriﬁed using

column chromatography on silica gel. Eluent CHCl₃-MeOH satu-

rated with NH₃(10:1 and 5:1).

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 0.95e1.08 (m, 2 H), 1.12e1.31

(m, 3 H), 1.53e1.88 (m, 8 H), 2.86 (dd, J ¼ 13.08, 7.34 Hz, 1 H), 2.98

(dd, J ¼ 13.14, 5.20 Hz, 1 H), 3.44 (s, 2 H), 3.73 (d, J ¼ 6.36 Hz, 2 H),

4.59 (d, J ¼ 5.26 Hz, 2 H), 4.87e4.99 (m, 1 H), 5.48 (t, J ¼ 5.56 Hz,

1 H), 6.84 (d, J ¼ 8.44 Hz, 2 H), 7.17 (d, J ¼ 8.44 Hz, 2 H), 7.52 (s, 1 H),

8.64 (d, J ¼ 7.82 Hz, 1 H).

12(fR), NBD-14294: M ¼ 516 mg. Yield ¼ 35% (over two steps).

rt ¼ 1.109 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 351 Da.

12(fS), NBD-14293: M ¼ 423 mg. Yield ¼ 36% (over two steps).

rt ¼ 1.109 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 351 Da.

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 25.3 (2 C), 26.1, 29.3 (2 C),

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 1.72 (br. s., 2 H), 3.02 (dd,

37.1, 41.3, 45.9, 54.4, 55.7, 72.7, 114.2, 127.9, 130.0, 139.1, 139.9, 157.5,

170.7, 171.6.

J ¼ 13.25, 7.99 Hz, 1 H), 3.15 (dd, J ¼ 13.32, 5.33 Hz, 1 H), 4.54 (d,

J ¼ 4.80 Hz, 2 H), 5.24 (br. s., 1 H), 5.28e5.34 (m, 1 H), 7.07 (dd,

J ¼ 8.53, 1.90 Hz, 1 H), 7.29 (s, 1 H), 7.35 (s, 1 H), 7.44 (d, J ¼ 1.37 Hz,

1 H), 7.69 (d, J ¼ 8.53 Hz, 1 H), 9.03 (br. s., 1 H), 11.80 (br. s., 1 H).

HRMS (ESI): m/z calcd for C₂₁H₃₀N₃O₃S [MþH]^þ404.2002,

found 404.2002.

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 45.7, 54.8, 59.7, 103.6, 111.7,

3.3.22. N-(2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)

ethyl)-1H-indole-2-carboxamide (10)

114.1, 120.3, 123.3, 125.8, 128.1, 132.1, 136.8, 157.6, 161.1, 171.8.

HRMS (ESI): m/z calcd for C₁₅H₁₆ClN₄O₂S [M ꢀ H]^-351.0677,

found 351.0681.

Compounds 10(fR), NBD-14032 and 10(fS), NBD-14033 were

obtained following the general procedure A and B from amine

1a(fR), 1a(fS), and acid 10b. Compounds were puriﬁed using col-

umn chromatography on silica gel. Eluent CHCl₃-MeOH saturated

with NH₃(10:1 and 5:1).

3.3.25. Allyl allyl(2-amino-2-(thiazol-2-yl)ethyl)carbamate (13c)

The thiazole (2.99 mL, 3.58 g, 42.1 mmol, 1.5 equiv.) was dis-

solved in THF (42 mL) and cooled to ꢀ78 ^ꢂC. At this temperature, n-

BuLi (2.5 M in hexane, 16.84 mL, 1.5 equiv.) was added dropwise

under a nitrogen atmosphere. The reaction mixture was stirred for

20 min at ꢀ78 ^ꢂC, and appropriate R- or S-imine (8.04 g, 28.1 mmol,

1 equiv.) was added dropwise as a solution in THF (1 M, 28 mL). The

reaction mixture was slowly (~1 h) warmed to 0 ^ꢂC and poured into

water (150 mL). The organic layer was separated, and water was

extracted with CH₂Cl₂(3 ꢁ 100 mL). Combined organic phases were

washed with brine (100 mL), dried over anhydrous Na₂SO₄, ﬁltered,

and evaporated to give a brown oil which was puriﬁed by ﬂash

column chromatography (eluent: hexane-EtOAc, 3:1 /1:1 / 0:1).

This product was used without analysis.

To a solution of the protected compound from the previous step

in MeOH (50 ml), 1 M solution of HCl in MeOH (100 ml) was added

in one portion. The mixture was stirred for 2e3 h. After that, sol-

vent was evaporated, and the residue was dissolved in water

(50 mL) and extracted from impurities with CH₂Cl₂(2 ꢁ 50 mL).

After that, solid K₂CO₃was added carefully (CO₂evolution!) to pH

10e12. The product was extracted from water with CH₂Cl₂

(4 ꢁ 50 mL), combined organic layers were dried over anhydrous

Na₂SO₄, ﬁltered, and evaporated to give pure amine.

10(fR), NBD-14032: M ¼ 58 mg. Yield ¼ 6% (over two steps).

rt ¼ 1.000 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 331 Da.

10(fS), NBD-14033: M ¼ 534 mg. Yield ¼ 32% (over two steps).

rt ¼ 1.170 min. Purity ¼ 99%. LC-MS: m/z [MþH]^þ¼ 331 Da.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 2.25 (s, 3 H), 3.02 (dd,

J ¼ 13.3, 8.1 Hz, 1 H), 3.13 (dd, J ¼ 13.1, 5.2 Hz, 1 H), 3.33 (br. s., 2 H),

4.52 (s, 2 H), 5.10e5.23 (m, 1 H), 5.37 (br. s., 1 H), 7.04 (t, J ¼ 7.5 Hz,

1 H), 7.19 (t, J ¼ 7.6 Hz, 1 H), 7.29 (s, 1 H), 7.43 (d, J ¼ 8.2 Hz, 1 H), 7.63

(d, J ¼ 7.9 Hz, 1 H), 8.91 (d, J ¼ 6.5 Hz, 1 H), 11.62 (s, 1 H).

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 14.9, 45.5, 54.5, 55.0, 103.5,

112.3, 119.8,121.6, 123.5,127.0,131.2,132.6,136.6,146.9,161.4, 169.0.

HRMS (ESI): m/z calcd for C₁₆H₁₉N₄O₂S [MþH]^þ331.1223, found

331.1221.

3.3.23. N-(2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)

ethyl)-5-chloro-1H-indole-2-carboxamide (11)

Compounds 11(fR), NBD-14030 and 11(fS), NBD-14031 were

obtained following the general procedure A and B from amine

1a(fR), 1a(fS), and acid 11b. Compounds were puriﬁed using col-

umn chromatography on silica gel. Eluent CHCl₃-MeOH saturated

with NH₃(10:1 and 5:1).

13c(fR): M ¼ 3.94 g. Yield (over two steps) ¼ 53%.

13c(fS): M ¼ 3.69 g. Yield (over two steps) ¼ 49%.

11(fR), NBD-14030: M ¼ 91 mg. Yield ¼ 10% (over two steps).

rt ¼ 1.137 min. Purity ¼ 98%. LC-MS: m/z [MþH]^þ¼ 365 Da.

11(fS), NBD-14031: M ¼ 616 mg. Yield ¼ 34% (over two steps).

rt ¼ 1.327 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 365 Da.

¹H NMR (CDCl₃, 400 MHz):

d

¼ 1.92 (br. s., 2 H), 3.49e3.96 (m,

4 H), 4.44e4.61 (m, 3 H), 5.03e5.20 (m, 3 H), 5.26 (d, J ¼ 17.24 Hz,

1 H), 5.73 (br. s., 1 H), 5.82e5.94 (m, 1 H), 7.24 (d, J ¼ 3.30 Hz, 1 H),

7.70 (d, J ¼ 3.18 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz):

d

¼ (50.6, 50.8), (53.1, 53.2), (53.4,

¹H NMR (DMSO‑d₆, 400 MHz):

J ¼ 13.2, 8.3 Hz 1 H), 3.15 (dd, J ¼ 13.1, 5.1 Hz, 1 H), 3.17 (br. s, 2H),

d

¼ 2.25 (s, 3 H), 3.03 (dd,

54.1), 66.3, (116.9, 117.4), (117.5, 117.9), 119.0, 132.8, 133.2, 142.8,

150.8, 175.3.

17

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European Journal of Medicinal Chemistry 224 (2021) 113681

3.3.26. N-(2-amino-1-(thiazol-2-yl)ethyl)-5-(4-chlorophenyl)-1H-

pyrrole-2-carboxamide (13)

2 H), 3.74e3.90 (m, 1 H), 6.68 (d, J ¼ 3.8 Hz, 1 H), 6.88 (d, J ¼ 3.8 Hz,

1 H), 7.53 (t, J ¼ 8.2 Hz, 1 H), 7.66 (dd, J ¼ 8.4, 1.6 Hz, 1 H), 7.84e7.97

(m, 2 H), 11.79 (br. s., 1 H). (One exchangeable proton signal is

missing).

Compounds 13(fR), NBD-14150 and 13(fS), NBD-14151 were

obtained following the general procedure A and B from amine

13c(fS), 13c(fR), and acid 13d [14]. The resulting compounds were

puriﬁed using column chromatography on silica gel. Eluent CHCl₃-

MeOH saturated with NH₃(10:1 and 5:1).

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 33.1, 45.2, 46.7, 108.3, 112.0,

112.4 (d, J¼23.0 Hz), 116.8 (d, J¼17.9 Hz), 121.6 (d, J¼3.3 Hz), 128.8,

130.7, 132.0 (d, J¼2.0 Hz), 133.1 (d, J¼7.9 Hz), 157.5 (d, J¼244.7 Hz),

159.3.

13(fR), NBD-14150: M ¼ 391 mg. Yield ¼ 27% (over two steps).

rt ¼ 1.262 min. Purity ¼ 97%. LC-MS: m/z [MþH]^þ¼ 347 Da.

13(fS), NBD-14151: M ¼ 420 mg. Yield ¼ 29% (over two steps).

rt ¼ 1.384 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 347 Da.

HRMS (ESI): m/z calcd for C₁₆H₁₈ClFN₃O [MþH]^þ322.1117, found

322.1122.

3.3.29. 5-(4-Chloro-3-ﬂuorophenyl)-N-(2,2,6,6-

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 1.73 (br. s., 2 H), 3.02 (dd,

tetramethylpiperidin-4-yl)-1H-pyrrole-2-carboxamide (16)

Compound 16, NBD-14095 was obtained following the general

procedure A from 2,2,6,6-tetramethylpiperidin-4-amine and acid

13d [14]. The resulting compound was puriﬁed using a column

chromatography on silica gel. Eluent: CHCl₃-MeOH saturated with

NH₃(10:1).

J ¼ 13.02, 7.76 Hz, 1 H), 3.16 (dd, J ¼ 13.20, 5.38 Hz, 1 H), 5.22e5.31

(m, 1 H), 6.65 (d, J ¼ 3.55 Hz, 1 H), 7.01 (d, J ¼ 3.55 Hz, 1 H), 7.42 (d,

J ¼ 8.31 Hz, 2 H), 7.58e7.63 (m, 1 H), 7.73e7.78 (m, 1 H), 7.84 (d,

J ¼ 8.31 Hz, 2 H), 8.58 (d, J ¼ 7.46 Hz, 1 H), 11.85 (br. s., 1 H).

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 45.7, 54.3, 107.6, 113.0, 119.6,

126.4, 127.4, 128.6, 130.7, 131.1, 133.8, 142.4, 160.5, 172.5.

HRMS (ESI): m/z calcd for C₁₆H₁₆ClN₄OS [M þ H]^-347.0728,

found 347.0725.

16, NBD-14095: M ¼ 182 mg. Yield ¼ 48%. rt ¼ 1.346 min.

Purity ¼ 94%. LC-MS: m/z [MþH]^þ¼ 378 Da.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 1.05 (s, 6 H), 1.12 (t,

J ¼ 12.3 Hz, 2 H), 1.17 (s, 6 H), 1.70 (dd, J ¼ 12.3, 3.6 Hz, 2 H), 3.33 (br.

s., 2 H), 4.17e4.31 (m, 1 H), 6.68 (d, J ¼ 2.9 Hz, 1 H), 6.85 (d,

J ¼ 3.6 Hz, 1 H), 7.54 (t, J ¼ 8.2 Hz, 1 H), 7.65 (dd, J ¼ 8.5, 1.8 Hz, 1 H),

7.77 (d, J ¼ 7.9 Hz, 1 H), 7.90 (dd, J ¼ 11.4, 1.9 Hz, 1 H), 11.73 (br. s.,

1 H). (One exchangeable proton signal is missing).

3.3.27. N-(2-aminoethyl)-5-(4-chloro-3-ﬂuorophenyl)-1H-pyrrole-

2-carboxamide hydrochloride (14)

Compound 14, NBD-14045 was obtained following the general

procedure A and Boc-deprotection (below) from tert-butyl (2-

aminoethyl)carbamate and acid 13d [14].

Boc-deprotection: To Boc-protected compound from the last step

was added 50 mL of 1 M HCl in methanol. The solution was stirred

for 2 h at room temperature, and then the solvent was evaporated.

The precipitate was triturated in Et₂O (50 mL) and ﬁltered. The

product was washed with Et₂O (2 ꢁ 50 mL) and CH₂Cl₂(2 ꢁ 50 mL)

and dried on the ﬁlter.

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 28.5, 35.0, 42.9, 45.5, 51.4,

108.5110.5, 113.0 (d, J ¼ 22.7 Hz), 119.5 (d, J ¼ 17.6 Hz), 121.1 (d,

J ¼ 3.7 Hz), 127.7, 131.1, 132.5 (d, J ¼ 8.1 Hz), 133.7, 158.5 (d,

J ¼ 248.1 Hz), 160.5.

HRMS (ESI) calcd for C₂₀H₂₆ClFN₃O [MþH]^þ378.1743, found

378.1736.

14, NBD-14045: M ¼ 217 mg. Yield ¼ 33% (over two steps).

rt ¼ 1.255 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 282 Da.

3.3.30. Tert-butyl 4-amino-4-cyanopiperidine-1-carboxylate (17b)

To a solution tert-butyl 4-oxopiperidine-1-carboxylate (20.0 g,

101 mmol, 1 equiv.) in MeOH-NH₃(60 mL) and aqueous NH₃

(40 mL), NaCN (5.42 g, 111 mmol, 1.1 equiv.) and NH₄Cl (6.45 g,

121 mmol, 1.2 equiv.) were added. The mixture was stirred for 4e5

days, then water (200 mL) was added and extracted with DCM

(3x150 mL). Combined organic layers were washed with brine

(200 mL) and dried over anhydrous Na₂SO₄, ﬁltered, and concen-

trated. The obtained crude product was used in the next step

without further puriﬁcation. The compound was obtained in

racemic form. M ¼ 21.47 g. Yield ¼ 95%.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 2.94e3.02 (m, 2 H),

3.45e3.54 (m, 2 H), 6.73 (s, 1 H), 6.90 (s, 1 H), 7.55 (t, J ¼ 8.2 Hz, 1 H),

7.72 (d, J ¼ 8.2 Hz, 1 H), 7.99 (d, J ¼ 11.6 Hz, 1 H), 8.04 (br. s., 3 H),

8.62 (t, J ¼ 4.8 Hz, 1 H), 12.07 (br. s., 1 H).

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 36.5, 38.8, 108.5, 112.6 (d,

J ¼ 22.7 Hz) 113.0, 116.9 (d, J¼17.6 Hz), 121.7 (d, J¼2.9 Hz), 128.3,

130.8, 133.0 (d, J¼7.3 Hz), 132.4, 157.5 (d, J¼244.4 Hz), 160.6.

HRMS (ESI): m/z calcd for C₁₃H₁₄ClFN₃O [MþH]^þ282.0804,

found 282.0804.

3.3.28. 5-(4-Chloro-3-ﬂuorophenyl)-N-(piperidin-4-yl)-1H-

pyrrole-2-carboxamide (15)

Compound 15, NBD-14094 was obtained following the general

procedure A and Boc-deprotection (below) from tert-butyl 4-

aminopiperidine-1-carboxylate and acid 13d [14].

¹H NMR (CDCl₃, 400 MHz):

d

¼ 1.46 (s, 9 H), 1.58e1.68 (m, 2 H),

1.86e2.01 (m, 4 H), 3.11e3.24 (m, 2 H), 3.95 (br. s., 2 H).

¹³C NMR (CDCl₃, 100 MHz):

(2 C), 80.3, 123.2, 154.5.

d

¼ 28.5 (3 C), 31.1, 36.9 (2 C), 50.0

Boc-deprotection: To Boc-protected compound was added 50 mL

of 1 M HCl in methanol. The solution was stirred for 2 h at room

temperature, and then the solvent was evaporated. Then 10%

aqueous K₂CO₃solution (50 mL) was added, followed by the

addition of CH₂Cl₂(100 mL). The layers were separated, and the

aqueous layer was extracted with CH₂Cl₂(2 ꢁ 50 mL). The com-

bined organic phases were dried over anhydrous Na₂SO₄and

evaporated. The compound was puriﬁed using column chroma-

tography on silica gel. Eluent: CHCl₃-MeOH saturated with NH₃

(10:1).

3.3.31. Tert-butyl 4-((tert-butoxycarbonyl)amino)-4-

cyanopiperidine-1-carboxylate (17c)

Compound 17b (23.13 g, 103 mmol, 1 equiv.) was dissolved in

THF (255 mL), then Boc₂O (22.38 g, 103 mmol, 1 equiv.) was added

in a few portions. The resulting mixture was stirred at room tem-

perature for 3 days. The solvent was evaporated, and the crude

product was puriﬁed by ﬂash chromatography on silica gel (eluent:

hexane-EtOAc, 5:1 / 1:1). M ¼ 25.39 g. Yield ¼ 76%.

¹H NMR (CDCl₃, 400 MHz):

d

¼ 1.45 (s, 9 H), 1.48 (s, 9 H),

15, NBD-14094: M ¼ 273 mg. Yield ¼ 64% (over two steps).

1.69e1.80 (m, 2 H), 2.32 (d, J ¼ 12.23 Hz, 2 H), 3.19e3.29 (m, 2 H),

rt ¼ 1.370 min. Purity ¼ 96%. LC-MS: m/z [MþH]^þ¼ 322 Da.

3.93 (d, J ¼ 13.57 Hz, 2 H), 4.93 (br. s., 1 H).

¹H NMR (DMSO‑d₆, 400 MHz):

1.75 (d, J ¼ 10.1 Hz, 2 H), 2.50e2.61 (m, 2 H), 2.97 (d, J ¼ 11.7 Hz,

d

¼ 1.39 (qd, J ¼ 11.8, 3.8 Hz, 2 H),

¹³C NMR (CDCl₃, 100 MHz):

d

¼ 28.2 (3 C), 28.3 (3 C), 34.9 (2 C),

39.8 (br., 2 C), 50.6, 80.2, 81.5 (br.), 119.2, 154.0 (br.), 154.3.

18

I.R. Iusupov, F. Curreli, E.A. Spiridonov et al.

European Journal of Medicinal Chemistry 224 (2021) 113681

3.3.32. Tert-butyl 4-((tert-butoxycarbonyl)amino)-4-

3.3.35. Tert-butyl 4-((tert-butoxycarbonyl)amino)-4-(4-

carbamothioylpiperidine-1-carboxylate (17d)

(hydroxymethyl)thiazol-2-yl)piperidine-1-carboxylate (17g)

To a solution of appropriate cyanopiperidine 17c (12.35 g,

38.0 mmol, 1 equiv.) in EtOH (380 mL), NaSH trihydrate (41.75 g,

380 mmol, 10 equiv.), and NH₄Cl (20.31 g, 380 mmol, 10 equiv.)

were added. The resulting mixture was stirred at room temperature

for 2 days and then poured into water (1000 mL). The resulting

solution was extracted with CH₂Cl₂(3 ꢁ 200 mL). The combined

organic layers were washed with water (200 mL) and brine

(200 mL), dried over anhydrous Na₂SO₄, ﬁltered, and evaporated to

dryness. The crude product was used in the next step without

further puriﬁcation. M ¼ 13.43 g. Yield ¼ 98%.

Compound 17f (14.37 g, 32.5 mmol, 1 equiv.) was dissolved in

EtOH (325 mL), then NaBH₄(12.37 g, 325 mmol, 10 equiv.) was

carefully added portion-wise. The resulting mixture was stirred at

room temperature for 15 min. Then the solution was reﬂuxed for

12e15 h, and the solvent evaporated to half volume and poured

into saturated NH₄Cl solution (500 mL), and extracted with CH₂Cl₂

(4 ꢁ 150 mL). The combined organic layers were dried over anhy-

drous Na₂SO₄, ﬁltered, and evaporated. The crude product was

puriﬁed by ﬂash chromatography on silica gel (eluent: hexane-

EtOAc, 5:1 / 1:1). M ¼ 3.98 g. Yield ¼ 30%.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 1.37 (s, 9 H), 1.38 (s, 9 H),

¹H NMR (CDCl₃, 400 MHz):

d

¼ 1.32 (br. s., 9 H), 1.39 (s, 9 H),

1.84e2.16 (m, 4 H), 2.85 (br. s., 2 H), 3.77 (d, J ¼ 8.44 Hz, 2 H), 6.98

1.99e2.38 (m, 4 H), 3.05 (br. s., 2 H), 3.86 (d, J ¼ 10.15 Hz, 3 H), 4.61

(br. s., 1 H), 8.69 (br. s., 1 H), 9.61 (br. s., 1 H).

(s, 2 H), 5.43 (br. s., 1 H), 7.04 (s, 1 H).

¹³C NMR (DMSO‑d₆, 100 MHz):

(br., 2 C), 39.8 (br.,2 C), 61.7, 78.3 (br.), 78.6, 153.6 (br.), 153.8, 154.2.

d

¼ 28.1 (3 C), 28.2 (3 C), 33.2

¹³C NMR (CDCl₃, 100 MHz):

36.0 (br.), 38.8 (br.), 39.8 (br.), 56.9, 60.7, 79.7, 80.0 (br.), 114.1, 154.4

(br.), 154.6, 156.3, 177.8.

d

¼ 28.3 (3 C), 28.4 (3 C), 35.1 (br.),

3.3.33. Ethyl 2-(1-(tert-butoxycarbonyl)-4-(N-(tert-

butoxycarbonyl)-2,2,2-triﬂuoroacetamido)piperidin-4-yl)thiazole-

4-carboxylate (17e)

3.3.36. (2-(4-aminopiperidin-4-yl)thiazol-4-yl)methanol

dihydrochloride (17h)

To a solution containing diBoc-protected compound 17g (3.98 g,

9.62 mmol) in MeOH (10 mL), 1 M HCl solution in MeOH (50 mL)

was added in one portion. The mixture was stirred at room tem-

perature for 4e5 h, and the solvent was evaporated to dryness. The

crude product was used in the next step without further puriﬁca-

tion. The desired product was obtained as a dihydrochloride.

M ¼ 2.7 g. Yield ¼ 98%.

Thioamide 17d (13.43 g, 37.4 mmol, 1 equiv.) was dissolved in

THF (374 mL), and to this stirred solution, KHCO₃(29.92 g,

299 mmol, 8 equiv.) and ethyl bromopyruvate (21.9 g, 112 mmol, 3

equiv.) were added at 0 ^ꢂC. After stirring for 5 min at this temper-

ature, the reaction mixture was warmed to 25 ^ꢂC, stirred for 30 h,

then concentrated. The residue was diluted in CH₂Cl₂(200 mL) and

washed with H₂O (250 mL), brine (250 mL), and dried over anhy-

drous Na₂SO₄. After the solution was concentrated, the residue was

taken up in THF (250 mL) and cooled to ꢀ10 ^ꢂC. Pyridine (30.1 mL,

374 mmol, 10 equiv.) and triﬂuoroacetic anhydride (25.96 mL,

187 mmol, 5 equiv.) were slowly added, and the solution was

allowed to stir for 2 h at 0 ^ꢂC. Then the reaction mixture was poured

into water (250 mL) and made slightly basic (pH ~ 8) with NaHCO₃.

The organic layer was separated; water was extracted with CH₂Cl₂

(3 ꢁ 100 mL). Combined organic phases were dried over anhydrous

Na₂SO₄, ﬁltered, and evaporated. The residue was subjected to ﬂash

chromatography on silica gel (eluent: hexane-EtOAc, 5:1 / 2:1) to

afford thiazole 17e. M ¼ 18.37 g. Yield ¼ 89%.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 2.36e2.47 (m, 2 H),

2.57e2.68 (m, 2 H), 2.91e3.02 (m, 2 H), 3.45e3.57 (m, 2 H), 4.58 (s,

2 H), 7.65 (s, 1 H), 9.36 (br. s., 3 H), 9.45 (br. s., 1 H), 9.79 (br. s., 1 H).

¹³C NMR (DMSO‑d₆, 100 MHz):

59.6, 117.6, 157.7, 167.3.

d

¼ 31.2 (2 C), 39.0 (2 C), 54.5,

3.3.37. Tert-butyl 4-amino-4-(4-(hydroxymethyl)thiazol-2-yl)

piperidine-1-carboxylate (17i)

Dihydrochloride 17h (2.2 g, 7.68 mmol,1 equiv.) was dissolved in

MeOH (77 mL), then DIPEA (13.4 mL, 76.8 mmol, 10 equiv.) was

added in one portion. The mixture was cooled to ꢀ40 to ꢀ30 ^ꢂC, and

Boc₂O (1.84 g, 8.46 mmol, 1.1 equiv.) was added dropwise as a so-

lution in CH₂Cl₂(10 mL). The reaction mixture was slowly warmed

to room temperature (~1e2 h) and stirred for 1 h. The resulting

solution was poured into 10% aqueous Na₂CO₃(100 mL), and it was

extracted with CH₂Cl₂(4 ꢁ 100 mL). Combined organic layers were

dried over anhydrous Na₂SO₄, ﬁltered, and evaporated. The crude

product was puriﬁed by ﬂash column chromatography on silica gel

(eluent: CH₂Cl₂-MeOH, 20:1 / 10:1). M ¼ 1.27 g. Yield ¼ 53%.

¹H NMR (CDCl₃, 400 MHz):

d

¼ 1.38 (t, J ¼ 7.15 Hz, 3 H), 1.46 (s,

9 H), 1.51 (s, 9 H), 2.32e2.48 (m, 2 H), 2.65e2.75 (m, 2 H), 3.34 (t,

J ¼ 10.64 Hz, 2 H), 3.78 (td, J ¼ 9.81, 3.97 Hz, 2 H), 4.38 (q,

J ¼ 7.09 Hz, 2 H), 8.14 (s, 1 H).

¹³C NMR (CDCl₃, 100 MHz):

(2 C), 39.7 (br., 2 C), 61.3, 65.6, 80.0, 87.3,115.3 (q, J¼288.3 Hz),127.9,

146.6, 150.4, 154.5, 161.1, 164.5 (q, J¼39.4 Hz), 173.1.

d

¼ 14.4, 27.4 (3 C), 28.4 (3 C), 35.5

¹H NMR (CDCl₃, 400 MHz):

d

¼ 1.46 (s, 9 H), 1.72 (d, J ¼ 13.45 Hz,

3.3.34. Methyl 2-(1-(tert-butoxycarbonyl)-4-((tert-

2 H), 2.07e2.17 (m, 2 H), 2.52 (br. s., 3 H), 3.37 (t, J ¼ 10.88 Hz, 2 H),

butoxycarbonyl)amino)piperidin-4-yl)thiazole-4-carboxylate (17f)

To a solution of 17e (18.37 g, 33.3 mmol) in MeOH (330 mL), a

MeONa solution in MeOH (1 M, 330 mL) was added. The mixture

was stirred at room temperature for 2 h, then poured into saturated

NH₄Cl solution (500 mL) and extracted with CH₂Cl₂(4 ꢁ 150 mL).

The combined organic layers were dried over anhydrous Na₂SO₄,

ﬁltered, and evaporated. The crude product was puriﬁed by ﬂash

chromatography on silica gel (eluent: hexane-EtOAc, 3:1 /1:1). By

treatment with sodium methylate, the corresponding methyl ester

was obtained. M ¼ 14.37 g. Yield ¼ 98%.

3.71e3.86 (m, 2 H), 4.71 (s, 2 H), 7.11 (s, 1 H).

¹³C NMR (CDCl₃, 100 MHz):

d

¼ 28.4 (3 C), 38.1 (2 C), 39.3 (br.),

40.1 (br.), 54.7, 60.6, 79.7, 114.5, 154.7, 156.5, 181.0.

3.3.38. N-(4-(4-(hydroxymethyl)thiazol-2-yl)piperidin-4-yl)-5-(4-

(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide (17)

DIPEA (0.704 mL, 4.05 mmol, 1 equiv.) was added to an appro-

priate acid 17k [14] (1.03 g, 4.05 mmol, 1 equiv.) followed by DMF

(10 mL) and then HBTU (1.54 g, 4.05 mmol, 1 equiv.). The resulting

solution was stirred for 5 min and added to a solution of appro-

priate amine 17i (1.27 g, 4.05 mmol, 1 equiv.) in DMF (10 mL) in one

portion. The reaction mixture was stirred overnight; DMF was

evaporated, and the residue was dissolved in CH₂Cl₂(100 mL) and

successively washed with 5% aqueous NaOH (50 mL) and 10% tar-

taric acid (50 mL) solutions. The organic layer was dried over

anhydrous Na₂SO₄, ﬁltered, evaporated. The crude product was

¹H NMR (CDCl₃, 400 MHz):

d

¼ 1.39 (br. s., 9 H), 1.45 (s, 9 H),

2.19e2.37 (m, 4 H), 3.12 (t, J ¼ 12.29 Hz, 2 H), 3.92 (s, 3 H), 3.99 (d,

J ¼ 13.69 Hz, 2 H), 5.14 (br. s., 1 H), 8.11 (s, 1 H).

¹³C NMR (CDCl₃, 100 MHz):

d

¼ 28.4 (3 C), 28.5 (3 C), 35.6 (br.,

2 C), 39.3 (br., 2 C), 52.5, 57.2, 80.0, 80.7 (br.), 127.7, 146.6, 154.4,

154.6, 162.0, 178.1 (br.).

19

I.R. Iusupov, F. Curreli, E.A. Spiridonov et al.

European Journal of Medicinal Chemistry 224 (2021) 113681

puriﬁed by ﬂash column chromatography on silica gel (eluent:

hexane-EtOAc, 3:1 / 1:1 / 0:1). (Note that after puriﬁcation, we

detected that product with N- and O-acylation was formed (Rf ¼ 0.3 in

hexane-EtOAc, 1:1). It was hydrolyzed with LiOH (10 equiv.) in THF-

MeOH-H₂O (1:1:1, 21 mL) under reﬂux for 2 h followed by acidiﬁca-

tion with aqueous HCl (12 M, 10 equiv.)).

To the Boc-protected compound was added 50 mL of 1 M HCl in

methanol and allowed 2 h in it. The solvent was evaporated, and

10% aqueous K₂CO₃(50 mL) was added, followed by CH₂Cl₂-EtOH

(~4:1, 50 mL) addition. The organic layer was separated, and the

aqueous layer was extracted with CH₂Cl₂-EtOH (~4:1, 4 ꢁ 50 mL).

The combined organic layers were dried over anhydrous Na₂SO₄,

ﬁltered, and concentrated. The crude product was puriﬁed by ﬂash

chromatography (twice) (eluent 1: CHCl₃-MeOH (saturated with

NH₃~7 M), 10:1 / 5:1, eluent 2: CH₂Cl₂-MeOH, 2:1 /1:1) afforded

the desired amine as a white solid.

product was extracted with CH₂Cl₂(3x100 mL). Combined organic

layers were dried over Na₂SO₄, ﬁltered, and evaporated. The residue

was puriﬁed using ﬂash chromatography on silica gel (eluent:

hexane-EtOAc 3:1 and pure EtOAc).

To a solution of protected thiazole from the previous step in

MeOH (50 ml), 1 M solution of HCl in MeOH (100 ml) was added in

one portion. The mixture was stirred for 2e3 h, and the solvent was

evaporated. The residue was recrystallized from methanol

(~20 mL). The precipitate was ﬁltered, quickly washed with 50 mL

of Et₂O-methanol (2:1) mixture, and quickly transferred under

vacuum (the product is hygroscopic). M ¼ 4.86 g. Yield (over two

steps) ¼ 59%.

S-enantiomer (fS) was synthesized by the same experimental

procedures. M ¼ 5.25 g. Yield (over two steps) ¼ 64%.

¹H NMR: (DMSO‑d₆, 400 MHz)

d

¼ 1.29e1.47 (m, 2 H), 1.50e1.64

(m, 1 H), 1.74 (d, J ¼ 13.2 Hz, 1 H), 1.77e1.90 (m, 2 H), 1.96 (dt,

J ¼ 13.6, 6.8 Hz,1 H), 2.69 (td, J ¼ 22.1,11.5 Hz, 2 H), 3.14 (dd, J ¼ 22.3,

12.3 Hz, 2 H), 4.56 (s, 2 H), 4.72 (d, J ¼ 5.0 Hz, 1 H), 7.51 (s, 1 H), 8.97

(br. s., 5 H), 9.08e9.21 (m, 1 H), 9.21e9.33 (m, 1 H).

17(NBD-14206): M ¼ 613 mg. Yield ¼ 34% (over two steps).

rt ¼ 1.340 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 451 Da.

¹H NMR (DMSO‑d₆, 400 MHz):

d

¼ 1.96e2.12 (m, 2 H), 2.51e2.57

(m, 2 H), 2.77e2.91 (m, 4 H), 3.47 (br.s.,1 H), 4.52 (s, 2 H), 5.22 (br. s.,

1 H), 6.78 (d, J ¼ 3.79 Hz, 1 H), 7.06 (d, J ¼ 3.79 Hz, 1 H), 7.23 (s, 1 H),

7.70 (d, J ¼ 8.44 Hz, 2 H), 8.03 (d, J ¼ 8.19 Hz, 2 H), 8.26 (s, 1 H), 12.01

(br. s., 1 H). (Two exchangeable proton signals are missing).

¹³C NMR: (DMSO‑d₆, 100 MHz)

42.7, 48.9, 59.6, 116.5, 157.9, 165.5.

d

¼ 27.7, 28.0, 29.6, 40.1, 42.7,

3.3.41. N-(1-(4-(hydroxymethyl)thiazol-2-yl)-2-(piperidin-4-yl)

ethyl)-5-(4-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

(18)

¹³C NMR (DMSO‑d₆, 100 MHz):

d

¼ 35.9 (2 C), 41.6 (2 C), 57.8,

59.9, 108.7, 113.3, 113.6, 124.4 (q, J¼272.2 Hz), 125.0 (2 C), 125.6 (q,

J¼3.7 Hz), 126.6 (q, J¼31.5 Hz, 2 C), 128.9, 133.1, 135.6, 156.9, 160.2,

178.2.

To a solution of salt 18c (fS or fR) (1.00 g, 2.85 mmol, 1 equiv.) in

DMF (10 mL), DIPEA (1.49 mL, 8.55 mmol, 3 equiv) was added. The

solution of Boc₂O (622 mg, 2.85 mmol, 1 equiv.) in DMF (5 mL) was

added dropwise. After 1 h in another ﬂask DIPEA (0.497 mL,

2.85 mmol, 1 equiv.) was added to an acid 17k [14] (728 mg,

2.85 mmol, 1equiv.) followed by DMF (8 mL) and then HBTU

(1.081 g, 2.85 mmol, 1 equiv.). A solution of activated acid was

added to a solution of protected amine. The reaction mixture was

stirred overnight. Then DMF was evaporated, and the residue was

dissolved in DCM (50 mL) and successively washed with 5%

aqueous NaOH and 10% tartaric acid or citric acid aqueous solutions

(50 mL). The organic layer was dried over Na₂SO₄, ﬁltered, evapo-

rated, and dry loaded on silica. Eluting with pure EtOAc gave pro-

tected compounds.

To Boc-protected compound was added 30 mL of 1 M HCl in

methanol. After 2 h, methanol was evaporated, and 10% aqueous

K₂CO₃(50 mL) was added, followed by CH₂Cl₂-EtOH (~4:1, 50 mL)

addition. The organic layer was separated, and the aqueous layer

was extracted with CH₂Cl₂-EtOH (~4:1, 4 ꢁ 50 mL). The combined

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated.

Puriﬁcation by ﬂash chromatography (twice) (eluent 1: CHCl₃-

MeOH (saturated with NH₃~7 M), 10:1 and 5:1, eluent 2: CH₂Cl₂-

MeOH, 4:1 and 2:1) afforded amine as a white solid.

HRMS (ESI): m/z calcd for C₂₁H₂₂F₃N₄O₂S [M þ H]^-451.1410,

found 451.1409.

3.3.39. Tert-butyl 4-(2-((tert-butylsulﬁnyl)imino)ethyl)piperidine-

1-carboxylate (18b)

Tert-butyl 4-(2-oxoethyl)piperidine-1-carboxylate (5.66 g,

25 mmol, 1 equiv.) was dissolved in CH₂Cl₂(25 mL) and (S)-(þ)-2-

methyl-2-propanesulﬁnamide (3.32 g, 27 mmol, 1.1 equiv.) was

added followed by anhydrous CuSO₄(9.94 g, 62 mmol, 2.5 equiv.)

addition. The reaction mixture was vigorously stirred for 1 day (at

this point, TLC indicated consumption of starting material). Hexane

(~100 mL) was added to the reaction mixture, and it was ﬁltered

through 1e2 cm plug of silica gel. The precipitate was washed with

the hexane-EtOAc mixture (1:1, 2x100 mL), and the ﬁltrate was

evaporated. The residue was used without further puriﬁcation.

M(fS) ¼ 7.56 g. Yield ¼ 92%.

Reaction with (R)-(þ)-2-methyl-2-propanesulﬁnamide was

performed in the same way.

M(fR) ¼ 7.73 g. Yield ¼ 92%.

¹H NMR: (CDCl₃, 400 MHz)

d

¼ 1.09e1.30 (m, 2H), 1.17 (s, 9 H),

1.43 (s, 9 H), 1.64e1.73 (m, 2 H), 1.80e1.97 (m, 1 H), 2.45 (t,

J ¼ 5.4 Hz, 2 H), 2.69 (t, J ¼ 10.3 Hz, 2 H), 3.87e4.31 (m, 2 H), 8.03 (t,

J ¼ 5.0 Hz, 1 H).

18(fR), NBD-14230: M ¼ 358 mg. Yield ¼ 26% (over three steps).

rt ¼ 1.444 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 479 Da.

18(fS), NBD-14228: M ¼ 554 mg. Yield ¼ 41% (over three steps).

rt ¼ 1.429 min. Purity ¼ 100%. LC-MS: m/z [MþH]^þ¼ 479 Da.

¹³C NMR: (CDCl₃, 100 MHz)

d

¼ 22.5, 28.5, 32.1, 33.7, 42.8, 43.9

(br.), 56.7, 79.5, 154.8, 168.3.

3.3.40. (2-(1-amino-2-(piperidin-4-yl)ethyl)thiazol-4-yl)methanol

trihydrochloride (18c)

Experimental procedures are given for the synthesis of R-

enantiomer (fR):

The appropriate thiazole (5.90 g, 25.7 mmol, 1.1 equiv.) was

dissolved in THF (26 mL). Under a nitrogen atmosphere, the solu-

tion was cooled to ꢀ78 ^ꢂC, and at this temperature n-BuLi (2.5 M in

hexane, 11.0 mL, 27.5 mmol, 1.2 equiv.) was added dropwise. The

reaction mixture was stirred for 10 min, and a solution of imine

18b(fR) (7.73 g, 23.4 mmol, 1 equiv.) in THF (26 mL) was added

dropwise. The resulting mixture was allowed to warm to room

temperature, stirred for 1 h, and diluted with water (~200 mL). The

¹H NMR: (DMSO, 400 MHz)

d

¼ 1.06e1.23 (m, 2 H), 1.43e1.53

(m, 1 H), 1.57 (d, J ¼ 12.7 Hz, 1 H), 1.71 (d, J ¼ 11.9 Hz, 1 H), 1.93 (t,

J ¼ 7.3 Hz, 2 H), 2.26e2.46 (m, 2 H), 2.90 (d, J ¼ 11.7 Hz, 2 H), 4.53 (s,

2 H), 5.42 (dd, J ¼ 15.3, 8.4 Hz, 1 H), 6.77 (d, J ¼ 3.8 Hz, 1 H), 7.03 (d,

J ¼ 3.8 Hz, 1 H), 7.27 (s, 1 H), 7.69 (d, J ¼ 8.4 Hz, 2 H), 8.03 (d,

J ¼ 8.2 Hz, 2 H), 8.79 (d, J ¼ 8.6 Hz, 1 H), 12.13 (br. s., 1 H). (Two

proton signals missing due to proton exchanging phenomena).

¹³C NMR: (DMSO, 100 MHz)

48.1, 59.8, 108.9, 113.1, 114.0, 124.4 (q, J ¼ 271.6 Hz), 125.1 (2C), 125.6

(q, J ¼ 3.7 Hz, 2C), 126.7 (q, J ¼ 31.7 Hz), 128.2, 133.4, 135.6, 157.7,

160.1, 174.2.

d

¼ 31.7, 32.6, 33.3, 41.5, 45.8, 45.9,

20

I.R. Iusupov, F. Curreli, E.A. Spiridonov et al.

European Journal of Medicinal Chemistry 224 (2021) 113681

HRMS (ESI): m/z calcd for C₂₃H₂₆F₃N₄O₂S [MþH]^þ479.1723,

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Acknowledgments

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22

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Design of gp120 HIV-1 entry inhibitors by scaffold hopping via isosteric replacements

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