Design, synthesis, and antiviral activity of a series of CD4-mimetic small-molecule HIV-1 entry inhibitors

Bioorg. Med. Chem. 32 (2021) 116000

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

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

Design, synthesis, and antiviral activity of a series of CD4-mimetic

small-molecule HIV-1 entry inhibitors

a

b

Francesca Curreli , Shahad Ahmed , Soﬁa M. Benedict Victor , Ildar R. Iusupov ,

b

Evgeny A. Spiridonov , Dmitry S. Belov , Andrea Altieri , Alexander V. Kurkin ,

a,*

Asim K. Debnath

a

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

b

EDASA Scientiﬁc, Scientiﬁc Park, Moscow State University, Leninskie Gory Bld. 75, 77-101b, 119992 Moscow, Russia

A R T I C L E I N F O

A B S T R A C T

Keywords:

We presented our continuing stride to optimize the second-generation NBD entry antagonist targeted to the

Phe43 cavity of HIV-1 gp120. We have synthesized thirty-eight new and novel analogs of NBD-14136, earlier

HIV-1

ENV-pseudovirus

Gp120 entry-antagonist

Cytotoxicity

designed based on a CH

2

OH “positional switch” hypothesis, and derived a comprehensive SAR. The antiviral data

conﬁrmed that the linear alcohol towards the “N” (C4) of the thiazole ring yielded more active inhibitors than

those towards the “S” (C5) of the thiazole ring. The best inhibitor, NBD-14273 (compound 13), showed both

improved antiviral activity and selectivity index (SI) against HIV-1HXB2 compared to NBD-14136. We also tested

NBD-14273 against a large panel of 50 HIV-1 Env-pseudotyped viruses representing clinical isolates of diverse

subtypes. The overall mean data indicate that antiviral potency against these isolates improved by ~3-fold, and

SI also improved ~3-fold compared to NBD-14136. This new and novel inhibitor is expected to pave the way for

further optimization to a more potent and clinically relevant inhibitor against HIV-1.

Broad-spectrum

Structure–activity relationship (SAR)

selectivity index (SI)

1

. Introduction

Human immunodeﬁciency virus type-1 (HIV-1) is the causative of

effective cure. Thus, the continued development of small-molecule

drugs with high potency against novel targets, but minimal side ef-

fects is imperative. The development of novel therapeutics will aid in

increasing the number of new drugs available and will extend the scope

of combination therapy.

the acquired immunodeﬁciency syndrome (AIDS). Based on the 2020

Global HIV Statistics from UNAIDS, 38 million people are living with

HIV globally, with about 1.7 million new infections in 2019. Since the

start of the epidemic, about 76 million people have become infected

with this deadly disease, and more than 32 million have lost their lives

due to AIDS-related illnesses.

Viral attachment and fusion to the host cell membrane are critical to

the ability of HIV-1 to enter host cells and initiate its life-cycle by uti-

lizing host cell machinery.¹Therefore, viral attachment and fusion

(often collectively termed “viral entry”) have been targets of new drug

discovery for years.¹Only two drugs currently on the market that

target the HIV-1 entry pathway. FUZEON® (enfuvirtide) targets the

envelope glycoprotein gp41,⁵and SELZENTRY® (maraviroc) targets

–4

However, advances in available therapeutics, particularly combina-

tion antiretroviral therapy (cART), signiﬁcantly improved the treatment

of HIV infection and facilitated the shift from high morbidity and mor-

tality to a manageable chronic disease. Despite this remarkable success,

current treatments suffer from several limitations, including (1) reliance

on daily adherence, (2) long-term use resulting in long-term toxicity, (3)

limited treatment options due to the development of drug resistance, (4)

high cost, and ﬁnally, (5) the non-curative nature of current treatments.

Further, despite the tremendous effort and investment for over a decade,

the availability of an effective vaccine or microbicide is not on the near

horizon, and signiﬁcant hurdles must be overcome to achieve an

,6

the host cell receptor CCR5 to prevent viral entry.⁷

–9

Even though HIV-1 gp120 is critical for viral entry and has been the

target of drug discovery efforts, no drugs that target gp120 have been

approved by the US FDA. BMS-663068, the most advanced inhibitor in

this class, is a prodrug of BMS-626529. The safety and efﬁcacy of BMS-

663068 were recently demonstrated in a Phase 2b clinical trial, and the

compound is currently undergoing Phase III clinical trials.

Our laboratory had focused on developing novel inhibitors targeted

*

Corresponding author.

E-mail address: adebnath@nybc.org (A.K. Debnath).

https://doi.org/10.1016/j.bmc.2021.116000

Received 3 December 2020; Accepted 31 December 2020

Available online 8 January 2021

0

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

to the Phe43 cavity of gp120 since 2005 when we ﬁrst reported the

discovery of NBD-556, the NBD-series CD4-mimic.¹⁰Unfortunately,

2. Results and discussions

ꢀ

+

NBD-556 was shown to enhance HIV-1 infection in CD4 -CCR5 cells

2.1. Design, anti-HIV-1 screening, and structure-activity relationships

(SARs)

and thus behaved as an HIV-1 entry agonist.¹

1,12

Since this ﬁnding, we

and others have endeavored to design CD4 mimics with not only higher

potency and lower toxicity but also that are devoid of this unwanted

In this study, we focused on one of the best lead NBD-14136 con-

agonist property and serve as HIV-1 entry antagonists.¹

2–22

3

taining a triﬂuoromethyl (CF ) group at the para position, which showed

Since the discovery of the small molecule CD4-mimic, NBD-556, we

systematically showed by x-ray crystallography that it binds to the

Phe43 cavity of the HIV-1 gp120.²³The major transformation of CD4-

mimic came through the design of NBD-11021. Based on the x-ray

structure of NBD-11021,¹²we further modiﬁed the molecules by

replacing the piperazine ring with a simple amine, such as NBD-14010.

The X-ray structure of NBD-14010 provided critical information on

modifying the thiazole ring substituents to achieve robust interactions

with the target protein.²⁴One such essential details lead us to postulate

the “Positional switch” hypothesis that resulted in more potent antivi-

rals, such as NBD-14136 (IC50 = 0.27 µM), NBD-14168 (IC50 = 0.28 µM),

an IC50 of 0.27 µM and CC50 of 42.4 µM resulting in an SI (where SI =

CC50/IC50) = 157. We synthesized a large set of compounds to improve

the antiviral activity and SI and derived a comprehensive SAR based on

3

the lead NBD-14136. In most cases, we kept the CF substituent at the

para position in the phenyl ring. We also decided to minimally alter the

substituents in the phenyl ring and make changes in the thiazole moiety

that showed improvement in antiviral activity and selectivity before.

Due to the presence of one chiral center, all compounds have two ste-

reoisomers (deﬁned as R and S in Table 1). We observed in our earlier

studies that in the majority of the cases, the S isomer had better antiviral

potency. Therefore, in the SAR, we will mostly focus on comparing the S

isomers. We have initially tested all the synthesized compounds against

HIV-1HXB2 in TZM-bl cells (Table 1) to identify the best active molecule

with higher SI to further test it against a large panel of HIV-1 ENV-

pseudotyped reference viruses from clinical isolates.

2

5

and NBD-14189 (IC50 = 0.089 µM) that we reported earlier (Figure 1).

Based on this success, we wanted to expand further the structur-

e–activity relationship (SAR) of these series of compounds with signiﬁ-

cant emphasis on compounds with the p-CF substituents, which yielded

3

the most active CD4-mimics as antivirals. In this report, we presented

our effort to expand the SAR studies of an extensive series of small

molecule CD4-mimics to optimize further this class of HIV-1 entry in-

hibitors that target the Phe43 cavity of envelope glycoprotein gp120 to

improve its antiviral activity and selectivity index (SI).

Compound 2 with a CF

OH at R positions yielded one of the most active compounds in this

series (IC50 = 0.19 µM and SI = 137). However, when we kept all groups

the same except changing CH OH to CH CH OH, the cytotoxicity

improved by about 1.7-fold, providing compound 13 with similar anti-

HIV-1 activity but much improved SI of 220. Moving the F to R in

3 2 4

at R and a ﬂuoro (F) at R position with

CH

2

6

2

1

compound 12 had no apparent effect on both antiviral activity and

cytotoxicity when compared to compound 2. We have explored a large

number of compounds keeping the substituents the same as in

Figure 1. “Positional switch” hypothesis indicating no interaction of the CH

2

OH group at position 5 in the thiazole ring in the x-ray structure of NBD-14010 bound to

HIV-1 gp120. When the CH

2

5

OH was switched to position 4 in the thiazole ring in NBD-14189, it forms H-bond (indicated by violet arrows using a glide docking pose)

2

with Met426 and Gly431.

2

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

Table 1

Anti-HIV-1 activity (IC50) of NBD compounds in a single-cycle assay against HIV-1 HXB2

.

b

New

No

Code

R

1

R

2

R

3

R

4

R

5

R

6

R

7

R

9

R

9

R

10

IC50

CC50

SI

(enantiomer)

a

NBD-14136 (R)

NBD-14250 (R)

H

CF

3

H

F

H

CH

H

2

OH

H

0.27 ± 02

0.61 ± 15

42.4 ± 1

26.8 ±

157

1

2

CH

2

OH

43.9

0

.25

NBD-14251 (S)

H

CF

3

H

F

H

CH

2

H

0.187 ±

25.7 ± 1.2

137

0

.01

3

4

5

6

7

8

9

NBD-14260 (R)

NBD-14261 (S)

NBD-14313 (S)

NBD-14314 (R)

NBD-14278-rac

NBD-14280-rac

NBD-14242 (S)

H

F

CN

H

F

H

CH

2

CH

2

CH

2

CH

2

CH

2

CH

2

CH

2

OH

H

>16

>82

–

H

>16

>82

–

CF

Cl

3

CF

H

3

H

4.2 ± 0.4

6.3 ± 1

1.6 ± 0.08

2.6 ± 0.4

0.15 ±

33.4 ± 2

37.6 ± 3.6

45.3 ± 1

47.7 ± 1.3

32.8 ± 1

7.9

5.9

28

18.3

218

3

H

CH

H

3

F

H

3

F

Cl

F

H

CH

2

OH

0

.001

1

0

1

2

3

4

5

NBD-14243 (R)

NBD-14258 (R)

NBD-14259 (S)

NBD-14273 (S)

NBD-14274 (R)

NBD-14287 (S)

F

Cl

F

H

F

H

CH

2

CH

2

OH

H

0.98 ± 0.01

0.51 ± 0.05

0.2 ± 0.02

0.18 ± 0.02

0.15 ± 0.01

0.55 ± 0.05

32.8 ± 0.5

24.2 ± 0.8

24.4 ± 0.7

39.6 ± 2

33.5

47.4

122

F

CF

3

H

F

H

F

220

F

23.4 ± 0.9

72.4 ± 3.1

156

H

CH(OH)

CH OH

CH(OH)

131.6

2

1

6

NBD-14288 (R)

F

CF

3

H

2.4 ± 0.1

56.3 ± 8.8

23.4

CH

2

CH

2

CH

2

H

OH

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

NBD-14289 (S)

NBD-14290 (R)

NBD-14303 (S)

NBD-14304 (R)

NBD-14271 (S)

NBD-14272 (R)

NBD-14275 (S)

NBD-14276 (R)

NBD-14267 (R)

NBD-14268 (S)

NBD-14325 (S)

NBD-14324 (R)

NBD-14329 (S)

NBD-14328 (R)

NBD-14246 (S)

F

CF

Cl

3

H

F

H

CH

2

CH

2

OH

H

CH

H

CH

H

0.25 ± 0.01

3.5 ± 0.2

6.2 ± 0.9

0.59 ± 0.1

Not soluble

2.9 ± 0.4

4.9 ± 0.9

2.3 ± 0.1

>10

22.6 ± 2.5

38 ± 1.3

98 ± 3.5

70.5 ± 4.5

–

90.4

10.8

15.8

119.4

–

14.3

4.8

2

H

CH(OH)CH

2

OH

H

CH(OH)CH

2

H

COOH

H

–

COOCH3

H

41.6 ± 1.3

23.4 ± 0.5

25.9 ± 0.3

>52

H

CONH

2

H

11.3

2

H

CH

H

2

OH

CH

H

3

0.54 ± 0.1

0.57 ± 0.1

0.15 ± 0.02

0.21 ± 0.04

0.12 ±

18.5 ± 0.2

14.1 ± 1.6

10.3 ± 0.5

10.4 ± 0.4

16 ± 1

34.2

24.7

20.6

49.5

133

2

CH

H

2

OH

H

2

CH

2

OH

3

0

.002

0.15 ±

.004

0.13 ±

.005

3

2

3

NBD-14247 (R)

NBD-14248 (S)

F

Cl

F

H

CH

H

CH

H

15.8 ± 0.5

17.5 ± 1

105

0

H

CF

3

H

134.6

0

3

4

5

6

7

NBD-14249 (R)

NBD-14262 (R)

NBD-14263 (S)

NBD-14225 (S)

H

CF

3

H

CH

H

CH

H

3

CH

H

3

H

1.3 ± 0.03

0.68 ± 0.12

0.23 ± 0.04

>13

19.9 ± 0.5

23.1 ± 0.5

25.1 ± 1

>61.9

15.3

34

CH

2

OH

H

OH

109

–

H

OCH

OH

2

CH(OH)

3

8

NBD-14229 (R)

H

CF

3

H

OCH

OH

2

H

2.1 ± 0.2

~80

~38

a

b

Data from Reference 25.

The reported IC50 and CC50 values represent the mean ± standard deviation (SD), n = 3.

compound 12 by increasing the complexity of the primary alcohol

Interestingly, we also observed that when we substituted the R

2

position

substituent at R

6

. In general, the cytotoxicity of the majority of the

with a hydrophilic substituent (CN), we virtually lost the antiviral po-

tency (Compound 4). It was, however, well-established that the Phe43

pocket in that region is hydrophobic. Therefore, the result was not

compounds improved, but the activity varied considerably. When the

chain length was increased in compound 17, the activity and cytotox-

icity remained similar to 12. The antiviral activity dropped considerably

2 1 3

surprising. A chloro (Cl) substituent at R and F in both R and R with

when R

6

had branched primary alcohols (compounds 15 and 19). When

CH CH OH at R (Compound 9) yielded one of the most active in-

2

6

we replaced the primary alcohol with an acid, the compounds became

insoluble; therefore, we could not perform the antiviral or cytotoxicity

assays. We also observed a considerable drop in antiviral activity when

hibitors with an IC50 of 0.15 µM and SI of 218. Interestingly, when we

added a methyl group at position R10 on the pyrrole ring, antiviral ac-

tivity improved remarkably (compounds 27–30). Unfortunately, the

cytotoxicity also was higher, making the SI values low. We also observed

R

6

had an amide or ester substituents. Interestingly, when we moved the

branched alcohol substituent to the R position, antiviral activity

dropped substantially (compound 19). It appears that bulkier alcohol

substituent in either R or R position was not well tolerated.

7

that when the primary amine had two methyl substituents at R

(compounds 31–34) and the CH OH group was at position R

antiviral activities were also improved substantially, but again these

8

and R

9

2

6

, the

6

7

3

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

additional methyl groups also made those molecules more cytotoxic

improvement in the anti-HIV-1 activity of our new generations of gp120

entry-antagonists against the control lab-adapted virus HIV-1HXB2 and a

(

CC50). However, when the CH

6), the antiviral activity dropped by about 2-fold, but cytotoxicity

improved to make SI > 100. In the same series, when we substituted R

2 7

OH group was moved to R (Compound

1

2,15,24,25

3

large panel of diverse HIV-1 Env-pseudotyped clinical isolates.

7

In this study, the anti-HIV-1 activity of NBD-14273 was evaluated

against a collection of 50 HIV-1 clinical isolates of diverse subtypes,

including primary, transmitted, and early founder HIV-1 isolates and a

selection of 12 recombinant HIV-1 clones (Table 2). All HIV-1 clones

tested use the CCR5 co-receptor for entry except for two dual-tropic

(CCR5/CXCR4) clones (NIH # 11563 and 11578). We compared the

antiviral activity of this compound with that of the previously described

inhibitor NBD-14136.²⁸NBD-14273 exhibited a broad-spectrum anti-

viral activity, as shown by its activity against all the clinical isolates

tested, regardless of the viral subtype or coreceptor usage. The anti-HIV-

1 activity of NBD-14273 was about 2.4–3.3-fold higher than the anti-

viral activity detected for NBD-14136, as demonstrated by their over-

all mean IC50 values and their antiviral activities against the different

HIV subtypes (Table 2). The overall mean IC50 value for NBD-14136 was

with branched alcohol (compounds 37 and 38), the antiviral potency

dropped substantially, but the cytotoxicity improved, although SI values

did not improve. Overall, the SAR analyses conﬁrmed that the antiviral

activity considerably improved by moving the primary alcohol groups to

6 7

R from R (as in NBD-14136). Both p-Cl and p-CF3 containing com-

pounds (compounds 9 and 13) showed high potency with high SI values.

The data in Table 1 demonstrated that NBD-14242 and NBD-14273

showed very similar antiviral activity and SI (IC50 of 0.15 and 0.18

µM; and 218 and 220, respectively). We selected NBD-14273 for further

study.

2

.2. NBD-14273 showed entry antagonist features

0

.39 ± 0.02 µM (with the SI of 108.7 and the IC50 values ranged from

NBD-556,¹⁰our ﬁrst-generation gp120-targeted compound, mimic

0

.19 to 0.79 µM), whereas the overall mean IC50 value determined for

CD4 by inducing a conformational change in gp120 and facilitating HIV

infection into CD4-negative cells that expressed the coreceptor

NBD-14273 was 0.135 ± 0.005 µM (with the SI of 293.3 and the IC50

values ranged from 0.11 to 0.22 µM). It is noteworthy to mention that

our earlier reported inhibitor NBD-14189, which showed the best ac-

tivity proﬁle (as low as 63 nM compared to 94 nM for NBD-14273), but

NBD-14273 demonstrated better SI (199.1 for NBD-14189 vs. 293 for

NBD-14273).²⁵In this study, NBD-14273 showed better antiviral ac-

tivity against all the viruses of different subtypes tested in this work. In

contrast, this compound exhibited a slightly better cytotoxicity proﬁle

1

3,26

CCR5.

Since then, we validated our new generation inhibitors by

conﬁrming that they do not possess this undesirable trait and work as

2

4,27

entry antagonists.

We selected NBD-14273, which exhibited the

best anti-HIV-1 activity and a higher Selectivity Index value (SI = 220).

CD4-negative Cf2TH-CCR5 cells were infected with the recombinant

CD4-dependant HIV-1ADA virus in the presence of escalating concen-

trations of NBD-14273 and NBD-556, which was used as a control. While

NBD-556 signiﬁcantly supported the infection of the Cf2Th-CCR5 cells,

NBD-14273 did not, indicating that this compound is an HIV-1 entry

antagonist (Figure 2). This data assured us of selecting this inhibitor for

further evaluation against a large and diverse panel of HIV-1 ENV-

pseudotyped clinical isolates.

(

CC50 of 39.6 ± 2 µM) than that for NBD-14136 (CC50 of 42.4 ± 1.0 µM),

which resulted in an improvement of the NBD-14273 SI values of 2.2–3-

fold with respect to NBD-14136. Moreover, NBD-14273 was poorly

active against the pseudovirus VSV-G, which was used here as a control,

suggesting that the inhibitory activities of these compounds are speciﬁc

to HIV-1. Additionally, the toxicity in the U87-CD4-CXCR4 cell line was

similar to what we detected in the other cell lines.

2

.3. Antiviral activities of NBD-14273 against a large and diverse panel

Furthermore, we evaluated the anti-HIV-1 activity of NBD-14273

against a panel of HIV-1 clones, which included a group of paired in-

fant and maternal clones. These HIV clones were isolated from chroni-

cally infected mothers and their respective infected infants and belonged

to subtypes A and D/A.²⁹it has been shown that the vertically trans-

mitted HIV-1 infant variants were more challenging to neutralize using

combinations of broadly neutralizing antibodies (bNAbs) 2G12, biz,

of HIV-1 Env-pseudotyped clinical isolates

Since we reported the identiﬁcation of NBD-556¹⁰as the ﬁrst

example of HIV-1 entry-inhibitor, we have shown consistent

2

F5, and 4E10.²⁹In this study, we found that NBD14273, as previously

2

8

shown for NBD-14136, equally neutralized both the infant and

maternal HIV-1 variants (Table 3), as shown by their overall mean of the

IC50 values and the IC50 means for infant and maternal viruses. Of note,

NBD-14273 was about 4-fold more effective than NBD-14136 against

both viral clones of the infant and maternal panel. These ﬁndings sug-

gest that NBD-14273 can neutralize both infant and maternal HIV-1

variants.

2

.4. Inhibitory activity of NBD-14273 against a large panel of FDA-

approved-drug-resistant viruses

We further evaluated the anti-viral activity of NBD-14273 against a

panel of drug-resistant viruses, consisting of 5 HIV-1 clones resistant to

the entry-inhibitor Enfuvirtide (T-20); 7 HIV-1 multi-drug-resistant

clones speciﬁcally, these are non-nucleoside reverse transcriptase in-

hibitor (NNRTI)-resistant viruses, which carry mutations that confer

resistance to both NNRTIs and nucleoside reverse transcriptase in-

hibitors (NRTIs); 3 HIV-1 clones resistant to the integrase inhibitor

Raltegravir; and 9 HIV-1 clones resistant to multiple protease inhibitors

Figure 2. Infectivity of CD4-negative Cf2Th

¡

CCR5 cells by CD4-

dependent HIV-1ADA CCf2Th CCR5 cells were infected with CD4-

.

ꢀ

dependent HIV-1ADA in the presence of NBD-14273 and NBD-556, which was

used as a control. The Relative virus infectivity indicates the ratio of the amount

of infection detected in the presence and absence of the compounds. Three

independent 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 CC50 values: for NBD-556, the CC50 was >

(

Table 4). As a reference, the activity of NBD-14273 was evaluated

against the wild type (WT) HIV-1NL4-3 clone, which was used to obtain

the drug-resistant viruses. For this assay, the TZM-bl cells were infected

with the WT HIV-1NL4-3 control virus or the drug-resistant viruses pre-

treated for 30 min with escalating concentrations of NBD-14273. NBD-

6

0, and for NBD-14273, it was 31.2 ± 1.4. All the values represent the mean ±

standard deviation.

4

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

Table 2

Neutralization Activity of gp120 entry-antagonists against a Panel of HIV-1 Env Pseudoviruses.

a

IC50 µM

c

Subtype

NIH #

ENVs

NBD-14136

0.46±0.06

0.31±0.07

0.44±0.05

0.26±0.04

0.41±0.03

0.62±0.08

0.29±0.01

0.41±0.03

0.34±0.02

0.6±0.005

0.58±0.05

0.41±0.01

0.36±0.06

0.33±0.01

0.39±0.01

0.52±0.07

0.51±0.06

0.52±0.02

0.36±0.04

0.27±0.01

0.41±0.06

0.39±0.02

0.37±0.07

0.32±0.02

0.22±0.03

0.32±0.08

0.58±0.06

0.25±0.02

0.58±0.01

0.29±0.02

0.36±0.08

NBD-14273

0.2±0.01

11887

11888

11890

11891

Q259ENV.W6

QB726.70M.ENV.C4

QF495.23M.ENV.A1

QF495.23M.ENV.A3

BG505-T332N

0.11±0.005

0.21±0.02

0.11±0.01

0.118±0.003

0.11±0.02

0.12±0.004

0.11±0.02

0.13±0.003

0.127±0.005

0.22±0.04

0.13±0.005

0.11±0.01

0.13±0.01

0.13±0.006

0.175±0.03

0.16±0.02

0.13±0.01

0.139±0.001

0.13±0.01

0.094±0.007

0.135±0.001

0.13±0.003

0.13±0.005

0.12±0.002

0.12±0.02

0.135±0.002

0.14±0.01

0.13±0.01

0.17±0.01

0.13±0.006

A

KNH1144

11901

11904

11905

11906

11591

11594

11595

QA790.204I.ENV.A4

QA790.204I.ENV.E2

QG393.60M.ENV.A1

QG393.60M.ENV.B7

CRF02_AG Clone 211

CRF02_AG clone 250

CRF02_AG clone 251

CRF02_AG clone 255

CRF02_AG clone 257

CRF13_cpx clone 258

CRF02_AG clone 266

CRF01_AE clone 269

B41

A/D

A2/D

A/G

11598

11599

11600

11602

AE

11603

11018

11022

11023

11036

11037

11038

11058

11560

11561

11562

11563

11572

QH0692, clone 42

PVO, clone 4

TRO, clone 11

RHPA4259 clone 7

THRO4156 clone 18

CAAN5342 clone A2

SC422661.8

B

1006_11.C3.1601

1054.TC4.1499

1056.TA11.1826

b

1058 11.B11.1550

9021_14.B2.4571

(

continued on next page)

5

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

Table 2 (continued)

b

1

1578

1307

1308

1309

1310

1312

1313

WEAUd15.410.5017

Du172, clone 17

Du422, clone 1

0.76±0.04

0.3±0.03

0.63±0.09

0.27±0.01

0.24±0.007

0.55±0.07

0.54±0.03

0.29±0.01

0.47±0.03

0.29±0.06

0.47±0.05

0.26±0.02

0.24±0.02

0.42±0.05

0.32±0.01

0.22±0.01

0.3±0.01

0.19±0.04

0.26±0.03

0.39±0.02

108.7

0.13±0.004

0.12±0.001

0.22±0.02

0.12±0.01

0.12±0.003

0.17±0.01

0.136±0.002

0.12±0.004

0.11±0.003

0.123±0.01

0.24±0.04

0.12±0.01

0.11±0.005

0.113±0.006

0.12±0.01

0.11±0.006

0.11±0.01

0.113±0.002

0.11±0.01

0.135±0.005

293.3

ZM197M.PB7, SVPC6

ZM214M.PL15, SVPC7

ZM249M.PL1, SVPC10

ZM53M.PB12, SVPC11

ZM109F.PB4

C

11314

11317

11502

11504

11506

11507

11908

11911

11912

11916

11918

CAP210.2.00.E8, SVPC17

HIV-16055-2, clone 3

HIV-16936-2, clone 21

HIV-25711-2, clone 4

HIV-225925-2, clone 22

QB099.391M.ENV.B1

QA013.70I.ENV.H1

QA013.70I.ENV.M12

QD435.100M.ENV.B5

QD435.100M.ENV.E1

CRF02_G clone 252

D

G

11596

Mean ± SEM (µM): Overall (n=50)

SI

Subtype A (n=6)

0.42±0.05

101

0.143±0.02

277

SI

Subtype Arec (n=12)

0.44±0.03

96.4

0.139±0.009

285

SI

Subtype B (n=14)

0.39±0.04

108.7

0.131±0.004

302.3

SI

Subtype C (n=13)

0.38±0.04

111.6

0.14±0.01

282.9

SI

Subtype D (n=4)

SI

0.26±0.03

163.1

0.11±0.002

360

>

20

IC50

11.9±0.5

33.5±0.6

d

Control

VSV-G

46.3±10.6

CC50

>

0.2 ≤0.5

>0.5

IC50 µM (Color Code)

≤0.2

a

The reported IC50 values represent the means ± standard deviations (n = 3).

b

c

R5X4-tropic virus all the rest are CCR5-tropic viruses.

28

Data previously published.

d

VSV-G was tested in U87-CD4-CCR5 cells

6

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

Table 3

Table 4

Neutralization activity of NBD-14273 against an HIV-1 panel of Paired Infant (B)

and Maternal (M) Env Molecular Clones.

Inhibitory activity of NBD-14273 against a large panel of FDA approved drug-

resistant viruses.

a

IC50 (µM)

NIH

Major mutations

NBD-

Fold

catalog#

14273

increase/

Sensitive

Subtype

NIH #

ENV

NBD-

NBD-14273

IC50

b

1

4136

(

µM)

A

11518-B

BG505.W6M.ENV.

C2

0.92 ±

0.16

0.22 ± 0.02

0.17 ± 0.05

0.18 ± 0.06

0.22 ± 0.07

0.13 ± 0.01

0.136 ± 0.02

0.24 ± 0.03

0.18 ± 0.03

NL4-3 wt (wild-

type)

#114

–

0.21 ±

0.04

–

A

11528-

M

MG505.W0M.

ENV.A2

0.93 ±

0.07

ENTRY

#9498

#9490

V38A, N42T

V38A

0.4 ±

0.08

1.9

(

Enfuvirtide)-

A

11519-B

B1206.W6P.ENV.

A1A

0.75 ±

0.06

resistant

0.44 ±

2.09

0

.02

A

11531-

M

MI206.W0M.ENV.

D1

0.7 ± 0.02

#

9496

9491

9489

V38E, N42S

N42T, N43K

D36G

0.31 ±

.03

0.62 ±

.07

0.34 ±

.06

1.48

0

A

11521-B

BJ613.W6M.ENV.

E1

0.8 ± 0.07

0.5 ± 0.05

2.95

0

A

11535-

M

MJ613.W0M.

ENV.A2

1.62

0

D/A

A

11524-B

BL035.W6M.ENV.

C1

0.52 ±

0.16

Multi-drug

#12227

#12229

K101P, K103N

L100I, K103N

K103N, Y181C

K101E, Y181V

Y181C, G190A

0.11 ±

0.006

Sensitive

1.86

(

NNRTI)-

11538-

M

ML035.W0M.

ENV.G2

0.59 ±

0.08

resistant

0.39 ±

0

.4

11525-B

BL274.W6M.ENV.

A3

0.63 ±

0.12

0.15 ± 0.020.136 ±

#

12231

12233

12237

12239

12241

0.12 ±

.006

0.71 ± ±

.02

0.3 ±

.02

Sensitive

3.38

0.01

0

A

11540-

M

ML274.W0M.

ENV.B1

0.66 ± 0.1

0

Mean ± SEM (µM): Overall (n = 10)

Infant (B) (n = 5)

0.7 ± 0.05

0.72 ±

0.176 ± 0.01

0.184 ± 0.02

1.4

0

.07

K101E, E138K,

Y181C

0.81 ± ±

0.08

3.85

Mother (M) (n = 5)

0.68 ±

.07

0.168 ± 0.016

0

K101E, G190S

0.26 ±

Sensitive

6.7

a

0

.01

The reported IC50 values represent the means ± standard deviations (n = 3).

b

28

#12243

#11847

#11850

L100I, M230L

G140S, Q148H

E92Q, N155H

N155H

1.4 ± ±

.05

Data previously published

0

Integrase

0.17 ±

0.02

Sensitive

1

4273 inhibited the WT HIV-1NL4-3, with an IC50 value of 0.21 µM. All

(Raltegravir-

resistant)

the T-20-resistant viruses were sensitive to NBD-14273; the IC50 values

we detected were similar to those determined for the control virus, WT

HIV-1NL4-3. Regarding the NNRTI-resistant viruses, also all the viral

clones were sensitive to NBD-14273. In some cases, we observed that the

drug-resistant viruses appeared to be more sensitive to the gp120 entry-

antagonist than the WT virus. The only exceptions were the HIV-1 clone

NIH # 12243, which showed a 6.7-fold increase of the IC50 with respect

to the IC50 determined for the WT HIV-1NL4-3 control virus. NIH #12233

and NIH #12239 clones showed a 3.38- and 3.85-fold increase of the

IC50, respectively. The Raltegravir-resistant viruses and the protease

inhibitor-resistant viruses were also sensitive to NBD-14273, as

demonstrated by their low IC50 values. In conclusion, NBD-14273 was

effective against all the drug-resistant viruses tested in this work, indi-

cating that this compound could potentially be used in combination with

other antiviral agents. To explain the low sensitivity of the HIV-1 clones,

NIH # 12243, NIH #12233, and NIH #12239 to NBD-14273 will require

further investigation.

0.11 ±

0

.1

#

11851

0.12 ±

.01

0

Multiple-PI-

resistant

#11800

11I, 32I, 33F, 46I,

47V, 54M, 58E,

0.1 ±

0.01

7

3S, 84V, 89V, 90M

#

11801

10F, 33F, 43T, 46L,

0.12 ±

Sensitive

5

9

4V, 82A, 84V,

0M

0.02

#11803

33F, 43T, 46I, 48V,

50V, 54S, 82A

0.38 ±

0.1

1.8

#

11804

11805

32I, 46I, 47V, 84V

0.24 ±

Sensitive

0

.02

48V, 53L, 54V,

0.16 ±

0.05

8

2A, 90M

32I, 33F, 47A, 82A,

0M

10F, 11I, 33F, 43T,

#11807

0.12 ±

0.03

9

#

11808

0.17 ±

0.03

4

8

6L, 54V, 73S, 82A,

4V, 89V, 90M

#

12465

12466

46I, 54V, 58E, 74P,

2L, 90M

32I, 33F, 43T, 46I,

0.13 ±

0.02

Sensitive

8

2

.5. gp120 entry-antagonists inhibited cell-to-cell HIV-1 transmission

0.11 ±

0.02

4

8

7V, 54M, 73S,

2A, 89V, 90M

Our earlier generation of gp120 entry-antagonists displays another

_c_r_i_t_i_c_a_l_f_e_a_t_u_r_e_:_t_h_e_a_b_i_l_i_t_y_t_o_i_n_h_i_b_i_t_c_e_l_l_-_t_o_-_c_e_l_l_H_I_V_-₁_t_r_a_n_s_m_i_s_s_i_o_n_.₂5,28

#12467

0.15 ±

Sensitive

0

.07

Cell-to-cell HIV-1 infection has been reported to be more efﬁcient than

a

3

0,31

Mutants were reported here as per the data obtained from https://www.

aidsreagent.org/ and associated references indicated in the Experimental

Section.

HIV-1 cell-free infection

as multiple viral particles can be trans-

mitted at the same time. Additionally, this type of infection is resistant to

some potent broadly neutralizing antibodies (bNAbs), including CD4

_b_i_n_d_i_n_g_s_i_t_e₍_C_D₄_b_s₎_a_n_t_i_b_o_d_i_e_s₃2-34 and NRTIs, but not to other anti-

b

The reported IC50 values represent the means ± standard deviations (n = 3).

retrovirals, including entry inhibitors, NNRTIs, and protease in-

_h_i_b_i_t_o_r_s_.₃5 Here, we evaluated the activity of NBD-14273 in the cell-to-

626529 was used as a control treatment drug. Our results (Table 5)

indicated that all the compounds tested in this assay, including BMS-

cell HIV-1 transmission setting and compared its activity to that of NBD-

6

26529, showed better activity against the CXCR4-tropic virus HIV-

1_I_I_I_Bthan against the CCR5-tropic virus HIV-1_A_D_A_.Furthermore, NBD-

4273 had slightly improved activity with respect to that we detected

for NBD-14136. Of note, the IC50 value for NBD-14273 calculated

1

4136, which has previously been reported to inhibit cell-to-cell HIV-1

2

5,28

transmission.

We used TZM-bl cells as acceptor cells and H9 cells

1

chronically infected with HIV-1IIIB (CXCR4-tropic), and MOLT-4 cells

chronically infected with HIV-1ADA (CCR5-tropic) as donor cells. BMS-

7

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

Table 5

The HIV-1 Subtype C Panel of Indian gp160 Env Clones HIV-16055-2

clone 3, HIV-16936-2 clone 21, HIV-25711-2 clone 4, and HIV-

225925-2 clone 22 were obtained through the NIH ARP from Drs. R.

Paranjape, S. Kulkarni and D. Monteﬁori.⁴⁴The panel of paired Infant

and maternal HIV-1 Env Molecular Clones were obtained through the

Inhibitory activity against Cell-to-Cell HIV transmission by NBD-14273.

a

Compound

IC50

( M)

μ

TZM-bl/H9-HIV-1IIIB

TZM-bl/Molt-HIV-1ADA

b

NBD-14136

NBD-14273

0.46 ± 0.2

0.89 ± 0.14

0.45 ± 0.01

~0.2

29

NIH ARP from Dr. J. Overbaugh. The Env pseudotyped genes of

0.39 ± 0.02

0.02 ± 0.005

BG505.T332N, KNH1144, and B41 were kindly provided by Dr. J. P.

Moore of the Weil Cornell Medical College, NY.

BMS-626529

a

The reported IC50 values represent the means ± standard deviation (SD), n =

The Env-deleted proviral backbone plasmids pNL4-3.Luc.R-.E-DNA

3

.

b

51,52

env

(

from Dr. N. Landau),

the pSG3Δ

DNA (from Drs. J. C. Kappes

28

Data previously published.

36,46

53

and X. Wu),

and the pNL4-3 (from Dr. Malcolm Martin) were

obtained through the NIH ARP Division of AIDS, NIAID, NIH. The

following molecular clones were also obtained through the NIH ARP,

against HIV-1ADA in the CCR5-tropic assay was 1-fold lower than the

IC50 values calculated for NBD-14136. When we compared the cell-cell

HIV-1 transmission inhibition of NBD-14273 with our earlier reported

most active inhibitor NBD-14189, it showed somewhat lower activity.²⁵

Division of AIDS, NIAID, NIH: the panel of Enfuvirtide (T-20) resistant

53,54

viruses from Trimeris, Inc.

; the panel Multi-Drug Resistant NNRTI

55

Infectious Clones from Dr. R. Shafer ; the Raltegravir-resistant infec-

5

6

tious molecular clones from Dr. R. Shafer and E. Reuman, M.S and the

multi-drug Protease inhibitor-resistant infectious clones from Dr. R.

Shafer.⁵⁷

3

. Conclusion

In summary, we have expanded the SAR of NBD-14136 generated

MLV gag-pol-expressing vector pVPack-GP, Env-expressing vector

pVPack-VSV-G, and a pFB-Luc vector were obtained from Stratagene (La

Jolla, CA).

through the “CH2OH” positional switch hypothesis reported earlier. A

systematic medicinal chemistry optimization and SAR study yielded

several new compounds with improved antiviral potency and SI. We

evaluated one of the best inhibitors, NBD-14273, against a large panel of

4

.2. Pseudovirus preparation

5

0 HIV-1 Env-pseudotyped viruses representing clinical isolates of

diverse subtypes. The data against this set of clinical isolates indicated

an improvement of antiviral potency and selectivity index by ~3-fold.

NBD-14273 also showed antiviral activity against a large panel of FDA-

approved-drug-resistant HIV viruses. The detailed data is expected to

pave the way to optimize this series further to a clinically relevant

candidate.

Pseudoviruses capable of single-cycle infection were prepared as

previously described.¹

3,15

Brieﬂy, 5 × 10 HEK293T cells were trans-

6

fected with an HIV-1 Env-expression plasmid and either the HIV-1 Env-

Δenv

deleted pro-viral backbone plasmid pSG3

or the pNL4-3.Luc.R-.E-

DNA, by using FuGENE6 (Promega). The control VSV-G pseudovirus

was prepared by transfecting the HEK293T cells with a combination of

the Env-expressing plasmid pVPack-VSV-G, the MLV gag-pol-expressing

plasmid pVPack-GP, the pFB-Luc plasmid by using FuGENE6.

Pseudovirus-containing supernatants were collected two days after

4

. Experiment

◦

4

.1. Cells and viruses

transfection, ﬁltered, tittered, and stored in aliquots at ꢀ 80 C.

TZM-bl cells,³⁶U87CD4 + CXCR4 + cells, HIV-1 IIIB, infected H9

37

4.3. Measurement of antiviral activity

Cells,³⁸and MOLT-4 CCR5 + Cells were obtained through the NIH

ARP. HEK 293T cells were purchased from ATCC. CD4-negative Cf2Th-

CCR5+ cells and Env expression vector pSVIIIenv-ADA were kindly

provided by Dr. J. G. Sodroski.⁴⁰HIV-1 Env molecular clone expression

vector pHXB2-env (X4) DNA was also obtained through the NIH ARP.⁴¹

HIV-1 Env molecular clones of gp160 genes for HIV-1 Env pseudovirus

production were obtained as follows: clones representing the standard

panels A (Q259ENV.W6, QB726.70 M.ENV.C4, QF495.23 M.ENV.A1,

QF495.23 M.ENV.A3), A/D, A2/D, D, and C (QB099.391 M.Env.B1)

were obtained through the NIH ARP from Dr. J. Overbaugh.⁴²^,⁴³The

HIV-1 Env molecular clones panel of subtype A/G, A/E, and G Env

clones were obtained through the NIH ARP from Drs. D. Ellenberger, B.

Li, M. Callahan, and S. Butera.⁴⁴The HIV-1 Env panel of standard

reference subtype B Env clones was obtained through the NIH ARP from

Drs. D. Monteﬁori, F. Gao and M. Li (PVO, clone 4 (SVPB11) TRO, Clone

39

4.3.1. Single-cycle infection assay in TZM-bl cells

The antiviral activity of the new generation of gp120 entry-

antagonists was evaluated in a single-cycle infection assay by infecting

TZM-bl cells with HIV-1 pseudotyped with the Env from the lab-adapted

CXCR4-tropic HIV-1HXB-2. Additionally, NBD-14136 and NBD-14273

were evaluated against a large group of HIV-1 pseudotyped with the

1

3,15

Env from the panel of clinical isolates as previously described.

4

Brieﬂy, TZM-bl cells were plated at 1 × 10 /well in a 96-well tissue

culture plate. Following overnight incubation, the cells were infected

with aliquots of HIV-1 pseudovirus pre-treated with graded concentra-

tions of the small molecules for 30 min. On the third day post-infection,

the cells were washed and lysed with 50 µl of lysis buffer (Promega). 20

µl of the lysates were transferred to a white plate and mixed with the

luciferase assay reagent (Promega). The luciferase activity was

measured immediately with a Tecan Spark reader, and the percent in-

hibition by the compounds and the IC50 (the half-maximal inhibitory

concentration) values were calculated using the GraphPad Prism

software.

1

1 (SVPB12), QH0692, clone 42 (SVPB6), SC422661, clone B (SVPB8));

from Drs. B. H. Hahn and J. F. Salazar-Gonzalez (pRHPA4259, clone 7

(

SVPB14)); from Drs. B. H. Hahn and D. L. Kothe (pTHRO4156 clone 18

4

5,46

SVPB15), pCAAN5342 clone A2 (SVPB19)).

p1058_11.B11.1550,

The subtype B clones

p1054.TC4.1499,

pWEAUd15.410.5017,

p1006_11.C3.1601, p1056.TA11.1826 and p9021_14.B2.4571 were

obtained through the NIH ARP from Drs. B. H. Hahn, B. F. Keele, and G.

M. Shaw.⁴⁷The subtype C HIV-1 reference panel of Env clones were also

obtained through the NIH ARP from Drs. D. Monteﬁori, F. Gao, S. A.

Karim, and G. Ramjee (Du172.17); from Drs. D. Monteﬁori, F. Gao, C.

Williamson, and S. A. Karim (Du422.1), from Drs. B. H. Hahn, Y. Li, and

J. F. Salazar-Gonzalez (ZM197M.PB7; ZM214M.PL15, ZM249M.PL1);

from Drs. E. Hunter and C. Derdeyn (ZM53M.PB12; ZM109F.PB4); from

4.3.2. Single-cycle infection assay in U87-CD4-CXCR4 cells

The antiviral activity of NBD-14136 and NBD-14273 was tested

against the control pseudovirus VSV-G in U87-CD4-CXCR4 cells. Brieﬂy,

U87-CD4-CXCR4 cells were plated in a 96-well tissue culture plate at 1

4

◦

× 10 /well and cultured at 37 C. Following overnight incubation, the

cells were infected with aliquots of pseudovirus pre-treated with graded

concentrations of the small molecules for 30 min. On the third day, post-

infection, the cells were washed and lysed with 40 µl of lysis buffer. The

lysates were then transferred to a white plate and mixed with the

Drs. L. Morris, K. Mlisana and D. Monteﬁori, (CAP210.2.00.E8).⁴

8–50

8

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

◦

luciferase assay reagent. The luciferase activity was immediately

measured to calculate the percent of inhibition and IC50 values by using

the GraphPad Prism software.

(Sigma) for 1 h at 37 C, washed and incubated with the TZM-bl cells in

the presence of escalating concentrations of compounds for 24 h. Then,

the cells were washed and lysed. The lysates were mixed with the

luciferase substrate, and the luciferase activity was measured to calcu-

late the percent of inhibition and IC50 values by using the GraphPad

Prism software.

4

.3.3. Assay in Cf2Th-CCR5 cells

3

CD4-negative Cf2Th-CCR5 cells were plated at 6 × 10 cells/well in a

◦

9

6-well tissue culture plate and incubated at 37 C. The following day,

the cells were infected with the recombinant CD4-dependent pseudo-

virus HIV-1ADA as previously described.¹¹Brieﬂy, aliquots of HIV-1ADA

pseudovirus pre-treated with graded concentrations of NBD-14273 and

NBD-556 for 30 min were added to the cells and cultured for 48 h. The

cells were then washed with PBS and lysed with 40 µl of cell lysis re-

agent. The lysates were mixed with the luciferase assay reagent, and the

luciferase activity was measured. For each compound, we calculated the

relative infection compared to the untreated control. The Relative virus

infectivity indicates 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.

4.6. Chemistry

For this study, all commercial reagents and solvents were used

without further puriﬁcation. Unless otherwise stated, all the described

reactions were performed in an air atmosphere. Reactions were moni-

tored by thin-layer chromatography (TLC) carried out on Merck TLC

Silica gel plates (60 F254), using a UV light for visualization and basic

aqueous potassium permanganate or iodine fumes as a developing

1

13

agent. H and C NMR spectra were recorded on a Bruker Avance 400

instrument with an operating frequency of 400 and 100 MHz, respec-

tively, and calibrated using residual not-deuterated chloroform (δH =

4

.3.4. Measurement of antiviral activity against drug-resistant viruses in

TZM-bl cells

We evaluated the antiviral activity of NBD-14273 against a panel of

7.28 ppm) and CDCl

3

(δC = 77.16 ppm) or not-deuterated DMSO (δH =

2.50 ppm) and DMSO‑d

6

(δC = 39.51 ppm) as internal references. NMR

data spectra abbreviations: s = singlet, d = doublet, t = triplet, q =

quartet, m = multiplet, br = broad. The purity of compounds was

determined via LCMS (Shimadzu LCMS-2010A) using three types of

detection systems such as EDAD, ELSD, and UV with purity cut-off at ≥

95%.

drug-resistant viruses by infecting TZM-bl cells. Brieﬂy, TZM-bl cells

4

were plated at 10 /well in a 96 well plate and cultured overnight. On the

following day, the HIV-1 drug-resistant viruses were pre-treated with

graded concentrations of the small molecules for 30 min and added to

the cells. Following 48 h incubation, the cells were washed and lysed.

The cellular lysates were mixed with the luciferase substrate, and the

luciferase activity was measured to calculate the percent inhibition by

the compounds and the IC50 values using the GraphPad Prism software,

as reported above.

4.6.1. General procedure A: For suzuki coupling

To a solution containing appropriate bromide (50 mmol, 1 equiv.),

(1-(tert-butoxycarbonyl)-1H-pyrrol-2-yl)boronic acid (50 mmol,

equiv.) in THF-H O (1:1, 100 mL), Na CO (100 mmol, 2 equiv.) and Pd

Ph P)Cl (1 mol. %) were added under a nitrogen atmosphere. The

mixture was stirred at reﬂux for 8–15 h (TLC-control). After cooling to

room temperature, water (50 mL) and CH Cl (50 mL) were added. The

organic layer was separated; the aqueous layer was extracted with

1

2

3

(

3

2

4

.4. Evaluation of cytotoxicity

2

.4.1. TZM-bl cells and U87-CD4-CXCR4 cells

The cytotoxicity of the gp120 entry-inhibitors in TZM-bl and U87-

CH

Na

2

Cl

2

(3x50 mL). The combined organic layers were dried over

4

, ﬁltered, and concentrated. Puriﬁcation by ﬂash chromatog-

CD4-CXCR4 cells was measured by using the colorimetric CellTiter

6® AQueous One Solution Cell Proliferation Assay (MTS) (Promega)

2

SO

9

raphy using hexane-EtOAc mixture as eluent afforded the desired com-

pound. Compound S1a-c were obtained following the general procedure

A (Scheme 1).

following the manufacturer’s instructions. Brieﬂy, the cells were seeded

◦

in a 96-well plate and cultured overnight at 37 C. The following day the

cells were cultured with 100 µl of the compounds at graded concentra-

tions and incubated for three days. The MTS reagent was added to the

4.6.2. Tert-butyl 2-(4-cyanophenyl)-1H-pyrrole-1-carboxylate (S1a)

Eluent: Hexane-EtOAc (from 20:1 to 10:1), Rf = 0.3 (10:1, Hexane-

◦

cells and incubated for 4 h at 37 C. The absorbance was recorded at 490

nm. The percent of cytotoxicity and the CC50 (the concentration for 50%

cytotoxicity) values were calculated as above.

EtOAc). Yield = 53%.

1

H NMR (CDCl

3

, 400 MHz): δ = 1.43 (s, 9H), 6.25–6.30 (m, 2H), 7.40

(

dd, J = 3.2, 1.9 Hz, 1H), 7.45–7.48 (m, 2H), 7.62–7.66 (m, 2H).

13

4

.4.2. Cf2Th-CCR5 cells

C NMR (CDCl

3

, 100 MHz): δ = 27.6 (3C), 84.3, 110.4, 111.0, 116.1,

The cytotoxicity of the small molecules in CD4-negative Cf2Th-CCR5

118.9, 123.9, 129.5 (2C), 131.3 (2C), 133.0, 138.7, 148.9.

cells was also measured with the colorimetric assay, as above. Brieﬂy,

◦

Cf2Th-CCR5 cells were plated in a 96-well plate and cultured at 37 C.

4.6.3. Tert-butyl 2-(2,4-bis(triﬂuoromethyl)phenyl)-1H-pyrrole-1-

carboxylate (S1b)

Following overnight incubation, the cells were cultured with 100 µl of

the compounds at graded concentrations and cultured for 48 h. The MTS

reagent was added to the cells, and 4 h later, the absorbance was

recorded at 490 nm. The percent of cytotoxicity and the CC50 values

were calculated as above.

Eluent: Hexane-EtOAc (from 1:0 to 20:1), Rf = 0.3–0.4 (20:1,

Hexane-EtOAc). Yield = 56%.

1

H NMR (CDCl

3

, 400 MHz): δ = 1.25 (s, 9H), 6.20–6.24 (m, 1H), 6.28

(t, J = 3.3 Hz, 1H), 7.43 (dd, J = 3.4, 1.8 Hz, 1H), 7.53 (d, J = 7.9 Hz,

1

H), 7.81 (d, J = 7.4 Hz, 1H), 7.97 (s, 1H).

13

4

.5. Cell-to-Cell HIV-1 transmission

C NMR (CDCl

3

, 100 MHz): δ = 27.4 (3C), 84.0, 110.7, 115.9, 122.4,

1

23.3 (q, J = 274.0 Hz), 123.1 (m), 123.6 (q, J = 272.2 Hz), 127.8 (q, J

The cell-to-cell HIV-1 transmission inhibition assay was performed as

= 3.7 Hz), 127.8, 130.6 (q, J = 33.5 Hz), 130.8 (q, J = 30.6 Hz), 133.6,

previously described⁵, with minor modiﬁcations. Brieﬂy, the indi-

8,59

138.0, 148.8.

4

cator TZM-bl cells (used as ‘acceptor’ cells) were plated at 10 /well in a

9

6 well plate 24 h before the assay. For the CXCR4-tropic assay, as

4.6.3.1. Tert-butyl 2-(2-ﬂuoro-4-(triﬂuoromethyl)phenyl)-1H-pyrrole-1-

‘

transmitting’ cells, we used H9 cells chronically infected with HIV-1IIIB

carboxylate (S1c). Eluent: Hexane-EtOAc (from 30:1 to 20:1), Rf = 0.5

3

at 2 × 10 cells/well. For the CCR5-tropic assay, we used MOLT-4/CCR5

(20:1, Hexane-EtOAc). Yield = 70%.

3

1

cells chronically infected with HIV-1ADA at 2 × 10 cells/well. The

H NMR (CDCl

3

, 400 MHz): δ = 1.41 (s, 9H), 6.28–6.33 (m, 2H),

transmitting cells were pre-treated with 200

μg/mL mitomycin C

7.30–7.38 (m, 1H), 7.42–7.47 (m, 2H), 7.46–7.52 (m, 1H).

9

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

Scheme 1. Synthesis of S4a-c.

, 100 MHz): δ = 27.6, 84.3, 111.0, 112.5 (dq, J =

¹³C NMR (CDCl

= 10.0 Hz), 113.6 (dq, J = 26.2, 3.9 Hz), 121.4 (d, J = 0.9 Hz), 121.6

(dq, J = 7.4, 3.7 Hz), 123.5 (d, J = 2.8 Hz), 123.6 (qd, J = 271.6, 2.4 Hz),

124.7 (d, J = 12.0 Hz), 126.5 (qd, J = 33.0, 8.3 Hz), 126.6 (d, J = 4.4

Hz), 157.3 (d, J = 249.0 Hz).

3

2

1

5.5, 3.7 Hz), 116.2, 120.8 (dq, J = 7.6, 3.7 Hz), 123.5 (dq, J = 272.2,

.6 Hz), 123.6, 126.8 (d, J = 14.9 Hz), 126.8, 131.3 (d, J = 3.3 Hz),

31.4 (qd, J = 33.2, 7.9 Hz), 149.0, 160.0 (d, J = 249.2 Hz).

4

.6.3.2. 4-(1H-Pyrrol-2-yl)benzonitrile (S2a). To a solution containing

4

.7. General procedure B: For acylation

S1a (10 g, 1 equiv.) in MeOH (20 mL), 1 M HCl solution in MeOH (100

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

Crude pyrrole from the previous step (1 equiv.) was dissolved in

perature for 7–8 h, and then the solvent was evaporated. Aqueous K

10% solution, 100 mL) was added carefully (CO evolution), and the

mixture was extracted with CH Cl (3x50 mL). The combined organic

layers were dried over Na SO , ﬁltered, and concentrated. The crude

2 3

CO

2 2

CH Cl (0.5 M solution), and pyridine (1.2 equiv.) was added, followed

(

2

by the dropwise addition of TFAA (1.2 equiv.). After completion of the

addition, the mixture was stirred for 2 h, and the solvent was evapo-

rated. The product was triturated in water, and the precipitate was

ﬁltered, washed with water twice, and dried on a ﬁlter. Compound S3a-

c were obtained following the general procedure B (Scheme 1).

2

4

product was used in the next step without puriﬁcation. M = 6.01 g.

Yield = 96%.

1

H NMR (CDCl

3

, 400 MHz): δ = 6.36 (dt, J = 3.5, 2.6 Hz, 1H), 6.69

(

ddd, J = J = 3.7, 2.6, 1.4 Hz, 1H), 6.97 (td, J = 2.7, 1.5 Hz, 1H),

4

.7.1. 4-(5-(2,2,2-Triﬂuoroacetyl)-1H-pyrrol-2-yl)benzonitrile (S3a)

7

1

4

.53–7.57 (m, 2H), 7.61–7.65 (m, 2H), 8.71 (br. s., 1H).

Yield = 79%.

13

C NMR (CDCl

3

, 100 MHz): δ = 108.7, 108.9, 111.1, 119.4, 121.1,

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 7.04 (dd, J = 4.2, 2.4 Hz, 1H),

23.8 (2C), 130.2, 132.9 (2C), 136.9.

7

1

.18–7.34 (m, 1H), 7.87 (d, J = 8.4 Hz, 2H), 8.14 (d, J = 8.4 Hz, 2H),

3.06 (br. s., 1H).

.6.3.3. 2-(2,4-Bis(triﬂuoromethyl)phenyl)-1H-pyrrole (S2b). To a solu-

13_C_N_M_R₍_D_M_S_O_‑_d

6

, 100 MHz): δ = 111.0, 112.3, 117.0 (q, J =

tion containing S1b (10 g, 1 equiv.) in CH

5.03 g, 132 mmol, 5 equiv.) was added dropwise on the water bath. The

mixture was stirred at room temperature overnight, and then the solvent

was evaporated. Aqueous K CO (10% solution, 100 mL) was added

carefully (CO evolution), and the mixture was extracted with CH2Cl2

3x50 mL). The combined organic layers were dried over Na SO

2 2

Cl (130 mL), TFA (10.1 mL,

2

1

89.9 Hz), 118.7, 123.0 (q, J = 3.7 Hz), 126.8 (2C), 126.9, 132.8 (2C),

34.0, 141.1, 168.3 (q, J = 35.2 Hz).

1

2

3

4

.7.2. 1-(5-(2,4-Bis(triﬂuoromethyl)phenyl)-1H-pyrrol-2-yl)-2,2,2-

2

triﬂuoroethanone (S3b)

(

2

4

,

Yield = 85%.

ﬁltered, and concentrated. Puriﬁcation by ﬂash chromatography using

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 6.56–6.62 (m, 1H), 7.31 (dt, J =

hexane-EtOAc mixture (from 30:1 to 10:1) as eluent (Rf = 0.2 in hexane-

4

.1, 2.1 Hz, 1H), 7.91 (d, J = 8.6 Hz, 1H), 8.14–8.26 (m, 2H), 13.17 (br.

EtOAc 10:1). M = 6.34 g. Yield = 86%.

s., 1H).

1

H NMR (CDCl

3

, 400 MHz): δ = 6.38–6.42 (m, 1H), 6.57–6.61 (m,

13_C_N_M_R₍_D_M_S_O_‑_d

6

, 100 MHz): δ = 114.1 (q, J = 2.4 Hz), 116.9 (q, J

289.9 Hz), 121.7 (q, J = 3.3 Hz), 123.0 (q, J = 273.9 Hz), 123.3 (qq, J

5.9, 4.0 Hz), 123.3 (q, J = 272.6 Hz), 126.1, 128.5 (q, J = 31.1 Hz),

1

H), 6.99–7.04 (m, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.83 (d, J = 8.3 Hz,

=

H), 8.04 (s, 1H), 8.62 (br. s., 1H).

1

3

C NMR (CDCl

3

, 100 MHz): δ = 110.1, 111.7, 121.0, 123.7 (q, J =

1

29.2 (q, J = 3.0 Hz), 130.0 (q, J = 33.2 Hz), 134.0, 134.1, 138.4, 168.8

2

1

72.0 Hz), 123.9 (q, J = 273.3 Hz), 124.1 (m), 126.7 (q, J = 31.1 Hz),

27.7, 128.8, (q, J = 4.0 Hz) 128.9 (q, J = 33.5 Hz), 131.9, 136.0.

(

q, J = 35.2 Hz).

4

.7.3. 2,2,2-Triﬂuoro-1-(5-(2-ﬂuoro-4-(triﬂuoromethyl)phenyl)-1H-

4

.6.3.4. 2-(2-Fluoro-4-(triﬂuoromethyl)phenyl)-1H-pyrrole (S2c). S2c

pyrrol-2-yl)ethanone (S3c)

Yield = 92%.

was synthesized by the same experimental procedures of S2b.

Eluent: Hexane-EtOAc (from 30:1 to 20:1), Rf = 0.4 (20:1, Hexane-

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 6.82 (t, J = 3.66 Hz, 1H),

EtOAc). Yield = 93%.

7

8

.13–7.36 (m, 1H), 7.64 (d, J = 8.27 Hz, 1H), 7.73 (d, J = 11.13 Hz, 1H),

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 6.18–6.27 (m, 1H), 6.72 (dd, J =

.21 (t, J = 7.95 Hz, 1H), 13.05 (br. s., 1H).

2

.29, 1.25 Hz, 1H), 6.96–7.06 (m, 1H), 7.56 (d, J = 8.25 Hz, 1H), 7.63

13

C NMR (DMSO‑d

6

, 100 MHz): δ = 113.6 (dq, J = 26.0, 3.8 Hz),

(

d, J = 11.86 Hz, 1H), 7.95 (t, J = 8.04 Hz, 1H), 11.54 (br. s., 1H).

1

14.4 (d, J = 10.7 Hz), 117.0 (q, J = 289.9 Hz), 121.5 (dq, J = 7.2, 3.5

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 109.7 (d, J = 1.7 Hz), 111.4 (d, J

Hz), 122.0 (d, J = 11.6 Hz), 122.3 (q, J = 3.0 Hz), 123.3 (qd, J = 277.2,

1

0

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

2

.4 Hz), 126.5, 130.1 (d, J = 2.6 Hz), 130.7 (qd, J = 33.4, 8.5 Hz), 135.4,

(Ph

3

P)Cl

2

(0.87 g; 1 mol. %), CuI (0.48 g; 2 mol. %) were added under

(

d, J = 1.8 Hz), 159.0 (d, J = 253.0 Hz), 168.7 (q, J = 35.2 Hz).

argon atmosphere. It was heated to 50–60 ^◦C, and the mixture was

stirred at this temperature for 6–8 h (TLC-control). Water (500 mL) was

added and extracted with hexane (3 × 150 mL). Combined extracts were

washed with water and dried with anhydrous sodium sulfate. The sol-

vent was removed by rotary evaporation, and the residue was puriﬁed

using ﬂash chromatography (eluent: from pure hexane to hexane-EtOAc,

4

.8. General procedure C: For haloform reaction

Appropriate triﬂuoroethanone (1 equiv.) was added to a solution of

2

NaOH (5 equiv.) in dioxane-H O mixture (1:1, 0.5 M solution). The

3

0:1, Rf = 0.6 in hexane). M = 30.1 g. Yield = 94%.

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. The resulting precipitate was ﬁltered, washed with

1

H NMR (CDCl

3

, 400 MHz): δ = 0.27 (s, 9H), 7.28 (d, J = 10.8 Hz,

1

H), 7.32 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H).

1

3

C NMR (CDCl

3

, 100 MHz): δ = -0.2, 99.0, 102.1 (d, J = 2.6 Hz),

H

2

O, and dried on a ﬁlter. Compound S4a-c were obtained following the

1

2

18.5 (qd, J = 33.2, 12.5 Hz), 120.1 (d, J = 22.1 Hz), 122.5 (q, J =

72.7 Hz), 127.2 (qd, J = 4.6, 2.2 Hz), 127.8 (d, J = 3.7 Hz), 129.4 (d, J

general procedure D (Scheme 1).

=

9.8 Hz), 159.5 (dq, J = 256.7, 2.0 Hz).

4

.8.1. 5-(4-Cyanophenyl)-1H-pyrrole-2-carboxylic acid (S4a)

Yield = 69%.

1

4

.8.5. 1-tert-Butyl 2-methyl 5-(3-ﬂuoro-4-(triﬂuoromethyl)phenyl)-3-

H NMR (DMSO‑d

6

, 400 MHz): δ = 6.75 (dd, J = 3.7, 2.5 Hz, 1H),

methyl-1H-pyrrole-1,2-dicarboxylate (S6)

6

.84 (dd, J = 3.8, 2.2 Hz, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.97 (d, J = 8.6

To a solution of S5 (20 g, 77 mmol, 1 equiv.) in DMF (400 mL),

triethylamine trihydroﬂuoride (3.72 g, 3.76 mL, 23 mmol, 0.3 equiv.)

was added, and the mixture was stirred for 1 h under argon atmosphere.

Then methyl (E)-2-(tert-butoxycarbonylamino)-3-iodo-but-2-enoate

Hz, 2H), 12.18 (br. s., 1H), 12.72 (br. s., 1H).

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 109.3, 116.6, 124.9 (2C), 125.4,

1

28.9, 129.9 (2C), 135.4, 135.6, 161.9, 167.2.

(

26.2 g, 77 mmol, 1 equiv.), Pd(Ph

3 2

P)Cl (2.7 g; 5 mol. %), CuI (1.46 g;

4

.8.2. 5-(2,4-Bis(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxylic acid

1

0 mol. %) and Cs CO (50.06 g; 154 mmol, 2 equiv.) were added under

2

3

(

S4b)

Yield = 71%.

argon atmosphere. It was heated to 70–80 ^◦C, and the mixture was

1

stirred at this temperature for 10–15 h (TLC-control). Water (800 mL)

H NMR (DMSO‑d

6

, 400 MHz): δ = 6.32–6.40 (m, 1H), 6.83 (dd, J =

was added and extracted with Et

2

O (3 × 200 mL). Combined extracts

SO

ﬁltered, and concentrated. Puriﬁcation by ﬂash chromatography

eluent: hexane-EtOAc from 20:1 to 5:1, Rf = 0.6 in hexane-EtOAc 5:1).

M = 12.73 g, Yield = 41%.

3

.7, 2.4 Hz, 1H), 7.83 (d, J = 8.6 Hz, 1H), 8.04–8.17 (m, 2H), 12.22 (br.

were washed with water and brine, dried over anhydrous Na

2

4

,

s., 1H), 12.55 (br. s., 1H).

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 111.9 (q, J = 3.0 Hz), 115.5,

(

1

23.2 (m), 123.4 (q, J = 273.9 Hz), 123.6 (q, J = 272.4 Hz), 125.3, 127.8

(

q, J = 30.8 Hz), 128.7 (q, J = 33.0 Hz), 128.9, 131.4, 134.0, 135.7,

1

H NMR (CDCl

3

, 400 MHz): δ = 1.46 (s, 9H), 2.30 (s, 3H), 3.89 (s,

1

62.0.

3

H), 6.14 (s, 1H), 7.25–7.33 (m, 2H), 7.61 (t, J = 7.7 Hz, 1H).

1

3

C NMR (CDCl

3

, 100 MHz): δ = 12.7, 27.4 (3C), 51.8, 85.8, 115.2,

4

.8.3. 5-(2-Fluoro-4-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxylic

1

2

1

2

17.3 (d, J = 21.9 Hz), 117.9 (qd, J = 33.3, 12.5 Hz), 122.6 (q, J =

72.2 Hz), 123.1, 124.6 (d, J = 3.7 Hz), 126.9 (qd, J = 4.6, 2.0 Hz),

30.0, 135.2 (d, J = 1.8 Hz), 138.1 (d, J = 8.9 Hz), 149.3, 159.3 (dq, J =

56.3, 2.0 Hz), 161.7.

acid (S4c)

Yield = 83%.

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 6.65–6.72 (m, 1H), 6.80–6.92 (m,

1

7

H), 7.61 (d, J = 8.27 Hz, 1H), 7.73 (d, J = 11.44 Hz, 1H), 8.20 (t, J =

.95 Hz, 1H), 12.19 (br. s., 1H), 12.64 (br. s., 1H).

1

3

4

.8.6. Methyl 5-(3-ﬂuoro-4-(triﬂuoromethyl)phenyl)-3-methyl-1H-

C NMR (DMSO‑d

6

, 100 MHz): δ = 112.6 (d, J = 10.6 Hz), 113.6

pyrrole-2-carboxylate (S7)

(

dq, J = 26.2, 3.7 Hz), 116.2, 121.5 (dq, J = 6.9, 3.2 Hz), 123.5 (qd, J =

2 2

To a solution of S6 (12.58 g, 31 mmol, 1 equiv.) in CH Cl (150 mL),

2

1

72.2, 2.8 Hz), 123.6 (d, J = 11.5 Hz), 125.7, 128.7 (d, J = 2.3 Hz),

28.7 (qd, J = 33.1, 8.7 Hz), 129.1 (d, J = 3.2 Hz), 158.4 (d, J = 250.9

TFA (17.87 g, 12 mL, 157 mmol, 5 equiv.) was added in one portion, and

the mixture was stirred overnight. Then the solvent was evaporated, and

Hz), 161.9.

1

0% aqueous K

2

CO

3

was added, and the mixture was extracted with

SO , ﬁltered, and

CH Cl (3x100 mL), dried over anhydrous Na

2

4

.8.4. ((3-Fluoro-4-(triﬂuoromethyl)phenyl)ethynyl)trimethylsilane (S5)

We synthesized S5–S8 based on Scheme 2. In a pressurized vessel

concentrated. The product was used without further puriﬁcation. M =

9

.28 g. Yield = 98%.

equipped with magnetic stirring bar containing solution of 4-bromo-2-

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 2.27 (s, 3H), 3.81 (s, 3H), 6.72 (d,

ﬂuoro-1-(triﬂuoromethyl)benzene (30 g; 123 mmol, 1 equiv.) in Et

3

N

J = 2.6 Hz, 1H), 7.70 (t, J = 8.1 Hz, 1H), 7.82 (d, J = 8.3 Hz, 1H), 8.02

(

250 mL), TMS-acetylene (24.25 g, 34.2 mL, 247 mmol, 2 equiv.), Pd

Scheme 2. Synthesis of S5-S8.

1

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

(

d, J = 12.9 Hz, 1H), 11.99 (br. s., 1H).

added in that sequence. The mixture was stirred at room temperature for

2–3 h (TLC-control), and then the solvent was evaporated. Crude

product was puriﬁed by ﬂash chromatography (eluent: from pure EtOAc

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 12.8, 51.1, 112.5, 112.8 (d, J =

2

1

2.7 Hz), 114.2 (qd, J = 32.4, 12.4 Hz), 121.0, 121.3, 122.8 (q, J =

71.5 Hz), 127.5 (q, J = 3.7 Hz), 128.4, 132.3, 137.9 (d, J = 9.0 Hz),

59.4 (d, J = 251.4 Hz), 161.2.

to CH

2

Cl

H NMR (CDCl

1.4, 3.8 Hz, 1H), 3.76–3.84 (m, 1H), 4.09 (br. s., 2H), 4.67 (s, 2H), 7.70

(s, 1H), 8.74 (s, 1H).

2

-MeOH 10:1, Rf = 0.3 in EtOAc). M = 32.2 g, Yield = 93%.

1

3

, 400 MHz): δ = 3.40–3.53 (m, 3H), 3.58 (dd, J =

1

4

.8.7. 5-(3-Fluoro-4-(triﬂuoromethyl)phenyl)-3-methyl-1H-pyrrole-2-

1

3

carboxylic acid (S8)

C NMR (CDCl

154.3.

3

, 100 MHz): δ = 63.6, 65.0, 70.7, 71.5, 135.4, 141.8,

To a solution of S7 (9.19 g, 31 mmol, 1 equiv.) in a mixture of EtOH-

2

H O (1:1, 120 mL), NaOH (2.44 g, 61 mmol, 2 equiv.) was added in one

portion, and the reaction mixture was stirred at reﬂux for 10–12 h (TLC-

control). Then the sodium-salt solution was acidiﬁed by the addition of

an equivalent amount of HCl (12 M, 5.08 mL, 2 equiv.), water (100 mL)

4.8.10. 5-((2,3-Bis((tert-butyldimethylsilyl)oxy)propoxy)methyl)thiazole

(S11)

To a solution of appropriate diol S10 (32.2 g, 170 mmol, 1 equiv.)

DMF (340 mL), imidazole (46.34 g, 681 mmol, 4 equiv.) was added in

one portion, followed by portionwise addition of TBSCl (102.57 g, 681

mmol, 4 equiv.). The reaction mixture was stirred overnight at 60–80 ˚C ,

cooled to the room temperature, diluted with water (680 mL), and

extracted with EtOAc (3 × 200 mL). The combined organic phases were

washed with water (200 mL), brine (200 mL), and dried over anhydrous

was added, and the precipitate was ﬁltered. M = 8.4 g. Yield = 96%.

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 2.28 (s, 3H), 6.72 (s, 1H), 7.70 (t,

J = 8.0 Hz, 1H), 7.83 (d, J = 8.1 Hz, 1H), 8.03 (d, J = 12.9 Hz, 1H),

1.91 (br. s., 1H), 12.53 (br. s., 1H).

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 12.8, 112.5, 112.6 (d, J = 22.7

Hz), 114.0 (qd, J = 32.1, 12.7 Hz), 120.9 (d, J = 3.0 Hz), 122.4, 122.9 (q,

J = 270.1 Hz), 127.6 (q, J = 3.5 Hz), 127.8, 131.6 (d, J = 1.8 Hz), 138.2

2 4

Na SO , ﬁltered and evaporated to give an oil, which was puriﬁed by

(

d, J = 9.4 Hz), 159.4 (dq, J = 249.2, 2.0 Hz), 162.5.

Compounds S9-S14 were synthesized as per Scheme 3.

ﬂash chromatography (eluent: hexane-EtOAc from 20:1 to 10:1, Rf = 0.4

in hexane-EtOAc 20:1). M = 59.95 g. Yield = 84%.

1

H NMR (CDCl

3

, 400 MHz): δ = 0.03–0.08 (m, 12H), 0.88 (s, 18H),

4

.8.8. 5-((Allyloxy)methyl)thiazole (S9)

3.46 (dd, J = 9.9, 5.7 Hz, 1H), 3.53–3.61 (m, 3H), 3.79–3.86 (m, 1H),

To a solution of appropriate alcohol (26.1 g, 227 mmol, 1 equiv.) in

4.77 (s, 2 H), 7.81 (s, 1H), 8.91 (s, 1H).

1

3

DMF (226 mL), NaH (40% in mineral oil, 9.97 g, 249 mmol, 1.1 equiv.)

C NMR (CDCl

3

, 100 MHz): δ = ꢀ 5.3, ꢀ 5.3, ꢀ 4.6, ꢀ 4.5, 18.3, 18.4,

◦

was added in several portions at 0 C. The mixture was stirred until gas

25.9 (3C), 26.0 (3C), 64.9, 65.4, 72.3, 72.7, 135.9, 141.9, 153.8.

evolution stopped. Then allyl bromide (23.5 mL, 32.91 g, 272 mmol, 1.2

equiv.) was added dropwise. The reaction mixture was stirred overnight

at room temperature. Water (500 mL) was added and extracted with

EtOAc (3 × 150 mL). Combined extracts were washed with water and

4.8.11. Allyl allyl(2-amino-2-(5-((3-((tert-butyldiphenylsilyl)oxy)-2-

hydroxypropoxy)methyl)thiazol-2-yl)ethyl)carbamate (S14)

Experimental procedures are given for the synthesis of R-enantiomer

(R):

brine, dried over Na

2 4

SO , ﬁltered, and concentrated. Puriﬁcation by

◦

distillation under reduced pressure (bp = 110–120 C, 10 torr). M =

The thiazole S11 (10.9 g, 26 mmol, 1 equiv.) was dissolved in THF

◦

2

8.34 g. Yield = 81%.

(26 mL) and cooled to ꢀ 78 C. At this temperature, n-BuLi (2.5 M in

1

H NMR (CDCl

3

, 400 MHz): δ = 4.01 (d, J = 5.7 Hz, 2H), 4.71 (s, 2H),

hexane, 10.96 mL, 1.05 equiv.) was added dropwise under a nitrogen

◦

5

1

.21 (d, J = 10.5 Hz, 1H), 5.29 (dd, J = 17.2, 1.6 Hz, 1H), 5.83–5.96 (m,

atmosphere. The reaction mixture was stirred for 20 min at ꢀ 78 C, and

H), 7.77 (s, 1H), 8.78 (s, 1H).

appropriate R-imine (S11a: 7.47 g, 26 mmol, 1 equiv.) was added

1

3

C NMR (CDCl

42.0, 153.9.

3

, 100 MHz): δ = 63.7, 71.0, 117.9, 134.0, 135.6,

dropwise as a solution in THF (1 M, 26 mL). The reaction mixture was

◦

1

slowly (~1h) warmed to 0 C and poured into water (150 mL). The

organic layer was separated, and water was extracted with CH

2

Cl

2

(3 ×

SO

4

.8.9. 3-(Thiazol-5-ylmethoxy)propane-1,2-diol (S10)

100 mL). Combined organic phases were dried over anhydrous Na

2

4

,

To a solution of alkene S9 (28.34 g, 183 mmol, 1 equiv.) in aceto-

ﬁltered, and evaporated to give a brown oil which was puriﬁed by col-

umn chromatography (eluent: hexane-EtOAc, gradient from 5:1 to 0:1,

Rf = 0.4 in hexane-EtOAc 1:1). This product (S12) was used without

ne–water (10:1, 360 mL), NMO monohydrate (37.02 g, 274 mmol, 1.5

equiv.) and potassium osmate (VI) dihydrate (0.67 g, 1 mol. %) were

Scheme 3. Synthesis of S9-S14 and S21.

1

2

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

further puriﬁcation and analysis.

mmol, 1 equiv.) and S-imine (S11a) (21.7 g, 75.6 mmol, 2 equiv.) in THF

◦

To a solution of protected thiazole from the previous step in MeOH

(38 mL) was cooled to 0 C and i-PrMgCl*LiCl solution (1 M in tetra-

(

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

hydrofuran, 78 mL, 78 mmol, 2 equiv.) was added maintaining the

◦

portion. The mixture was stirred for 2–3 h, and the solvent was evapo-

rated. The residue was dissolved in water (50 mL) and extracted with

temperature between 0 and +5 C. The reaction mixture was then stirred

◦

at 0 C for 1 h and allowed to warm to room temperature. The reaction

CH

evolution!) to the water phase at pH 10–12. Product was extracted from

water with CH Cl

(4 × 50 mL), combined organic layers were dried

over anhydrous Na SO

, ﬁltered, and evaporated to give diol S13. M =

2

Cl

2

(2 × 50 mL). After that, solid K

2

CO

3

was added carefully (CO

2

was quenched by the addition of aqueous ammonium chloride solution

(20% w/w, 0.5 L) and extracted with Et

2

O (3 × 100 mL). Combined

SO

2

organic layers were washed with brine (3 × 50 mL) dried over Na

2

4

2

4

and evaporated to dryness. The residue was dissolved in excess of 1 M

HCl n methanol (400 mL) and left for 2 days. Methanol was evaporated,

3

.02 g.

¹H NMR (CDCl

3

, 400 MHz): δ = 2.26 (br. s., 4H), 3.49–3.72 (m, 6H),

and saturated NaHCO

was extracted with CH

dried over anhydrous Na

was puriﬁed using column chromatography (eluent: hexane-EtOAc from

3

(100 mL) was added to the residue. The product

Cl

(3 × 50 mL). The combined organic layer was

SO and evaporated to dryness. The residue

3

4

2

.77–3.98 (m, 3H), 4.46 (t, J = 6.5 Hz, 1H), 4.60 (d, J = 5.3 Hz, 2H),

.70 (s, 2H), 5.08–5.24 (m, 3H), 5.30 (d, J = 17.2 Hz, 1H), 5.70–5.97 (m,

H), 7.58 (s, 1H).

2

4

Diol S13 (3.02 g, 8 mmol, 1 equiv.) was dissolved in CH

2

Cl

2

(81 mL),

1:1 to 0:1, and then CH

2

Cl

2

-MeOH 10:1, R

f

= 0.3–0.4 in EtOAc). M =

and imidazole (0.66 g, 10 mmol, 1.2 equiv.) was added in one portion,

followed by dropwise addition of TBDPSCl (2.33 mL, 2.46 g, 9 mmol, 1.1

equiv.). The reaction mixture was stirred overnight, diluted with water

7.74 g. Yield = 63%.

Reaction with R-imine (S11a) was performed in the same way. M =

6.22 g. Yield = 51%.

1

(

50 mL), and extracted with CH

2

Cl

2

(3 × 50 mL). The combined organic

SO . It was

H NMR (DMSO‑d

6

, 400 MHz): δ = 2.48 (br. s., 2H), 3.36–3.41 (m,

phases were washed with brine and dried over anhydrous Na

2

4

1H), 3.55–3.68 (m, 1H), 3.81 (s, 3H), 3.87 (d, J = 5.2 Hz, 2H), 4.32–4.54

(m, 3H), 5.04–5.18 (m, 3H), 5.25 (d, J = 17.3 Hz, 1H), 5.70–5.95 (m,

2H), 8.43 (s, 1H).

then ﬁltered and evaporated to give an oil, which was puriﬁed by ﬂash

chromatography (eluent: hexane-EtOAc, from 1:1 to 0:1 and then

CH

2

Cl

2

-MeOH 10:1, Rf = 0.3 in EtOAc). M = 3.75 g. Yield (over three

¹³C NMR (DMSO‑d

6

, 100 MHz): δ = 50.0, 51.9, 52.4, (52.7, 53.2),

steps) = 24%.

65.3, (116.0, 116.7), (116.5, 116.9), 129.2, 133.3, (133.8, 134.0),

145.7, (155.1, 155.8), 161.4, (177.9, 178.2).

S-enantiomer (S) was synthesized by the same experimental pro-

cedures. Yield (over three steps) = 17%.

1

H NMR (CDCl

3

, 400 MHz): δ = 1.06 (s, 9H), 1.84 (br. s., 2H), 2.52

4.8.13. Allyl allyl(2-amino-2-(4-carbamoylthiazol-2-yl)ethyl)carbamate

(S16)

(

br. s., 1H), 3.51–3.72 (m, 6H), 3.77–4.01 (m, 3H), 4.45 (t, J = 6.4 Hz,

H), 4.61 (d, J = 5.3 Hz, 2H), 4.67 (s, 2H), 5.08–5.19 (m, 2H), 5.21 (dd,

1

Thiazole S15 (1.5 g, 4.6 mmol) was dissolved in MeOH saturated

◦

J = 10.4, 1.2 Hz, 1H), 5.31 (dd, J = 17.2, 1.3 Hz, 1H), 5.71–5.98 (m,

with NH3 (~7M, 10 mL). It was heated to 70–80 C in a pressure vessel

2

H), 7.36–7.47 (m, 6H), 7.57 (s, 1H), 7.65 (d, J = 6.6 Hz, 4H).

on an oil bath, and the mixture was stirred at this temperature for 25–30

1

3

C NMR (CDCl

3

, 100 MHz): δ = 19.1, 26.7 (3C), (50.4, 50.6), (53.1,

h (TLC-control). Then it was cooled to the room temperature and

5

3.2), 64.6, 65.3, 66.1, 70.6, 70.9, (116.8, 117.2), (117.3, 117.7), 127.7

evaporated. The product was used without further puriﬁcation.

1

(

4C), 129.7 (2C), 132.7, 133.0 (2C), 133.1, 135.1, 135.4 (4C), 141.4,

155.8, 156.7), (175.5, 176.3).

H NMR (CDCl

3

, 400 MHz): δ = 1.94 (br. s., 2H), 3.49–3.71 (m, 2H),

3.75–3.98 (m, 2H), 4.42–4.63 (m, 3H), 5.05–5.33 (m, 4H), 5.66–5.97

Compounds S15, S16, and S21 were synthesized as per Scheme 4.

(m, 2H), 6.28 (br. s., 1H), 7.15 (br. s., 1H), 8.07 (s, 1H).

1

3

C NMR (CDCl

3

, 100 MHz): δ = 50.3, (52.3, 52.5), (53.4, 52.6),

4

.8.12. Methyl 2-(2-(allyl((allyloxy)carbonyl)amino)-1-aminoethyl)

65.9, (116.6, 117.0), (117.1, 117.4), 123.9, 132.2, 132.7, 149.3, (155.6,

156.4), 163.1, (174.9, 175.3).

thiazole-4-carboxylate (S15)

A solution of tert-butyl 2-bromothiazole-4-carboxylate (10.0 g, 37.9

We prepared S17-S20 and S22-S28 were prepared by following the

1) iPrMgCl*LiCl,

THF, -5°C

NH ₃(solution in MeOH)

2) S- or R-

S11a

3) HCl-MeOH

S15

S16

1) BuLi, THF, -78°C

2) S- or R-

S21a

2) HCl, MeOH

S21

Scheme 4. Synthesis of S15, S16 and S21.

1

3

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

methods reported previously.²⁵^,²⁷^,²⁸^,⁶⁰

4.11. General procedure F: For TBDPS- deprotection

4

.8.14. Allyl (1-amino-1-(4-(hydroxymethyl)thiazol-2-yl)-2-

TBDPS cleavage: To a solution of TBDPS-protected compound (1

equiv.) in THF (0.1 M), solution of TBAF trihydrate (1.1 equiv.) in THF

(0.1 M) was added in a one portion. The mixture was stirred for 1–2 h at

room temperature (TLC-control) and concentrated. Puriﬁcation by ﬂash

methylpropan-2-yl)carbamate (S21)

Experimental procedures for the synthesis of R-enantiomer (R):

The 4-(((tert-Butyldimethylsilyl)oxy)methyl)thiazole (20.02 g, 87

◦

mmol, 2.5 equiv.) was dissolved in THF (87 mL) and cooled to ꢀ 78 C.

chromatography (twice) (eluent 1: CHCl

3

-MeOH (saturated with

At this temperature, n-BuLi (2.5 M in hexane, 34.92 mL, 2.5 equiv.) was

NH3~7M), 10:1, 5:1, 3:1, eluent 2: CH Cl -MeOH, 4:1, 3:1, 2:1, 1:1)

2

added dropwise under a nitrogen atmosphere. The reaction mixture was

afforded amine as a slightly yellow or white solid.

◦

stirred for 20 min at ꢀ 78 C, and appropriate R-imine (S21a) (10.0 g,

Compounds 1–38 were synthesized as per Scheme 5.

3

5 mmol, 1 equiv.) was added dropwise as a solution in THF (1 M, 35

◦

mL). The reaction mixture was slowly (~1h) warmed to 0 C and poured

into water (200 mL). The organic layer was separated, and water was

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

4-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

extracted with CH

2

Cl

2

(4 × 100 mL). Combined organic phases were

SO4, ﬁltered, and evaporated to give a brown

Compounds 1 (NBD-14250) and 2 (NBD-14251) were obtained

following the general procedure D, and E from amine S17 and acid S4c

Compounds were puriﬁed using column chromatography on silica gel

dried over anhydrous Na

2

oil which was puriﬁed by column chromatography (eluent: hexane-

EtOAc, gradient from 5:1 to 0:1, Rf = 0.4 in hexane-EtOAc 1:1). This

product was used without analysis.

3 3

(twice). Eluent 1: CHCl -MeOH (saturated with NH ~7M), 10:1 and 5:1,

Eluent 2: CH Cl -MeOH, 4:1 and 2:1.

2

To a solution of protected thiazole from the previous step in MeOH

1 (NBD-14250); (R): M = 501 mg. Yield = 29% (over two steps). rt =

+

(

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

1.317 min. Purity = 100%. LC-MS: m/z [M + H] = 429 Da.

portion. The mixture was stirred for 2–3 h, and the solvent was evapo-

rated. The residue was dissolved in water (50 mL) and extracted with

2 (NBD-14251); (S): M = 432 mg. Yield = 25% (over two steps). rt =

+

1.325 min. Purity = 100%. LC-MS: m/z [M + H] = 429 Da.

1

CH

evolution) to the water phase at pH 10–12. Product was extracted from

water with CH Cl

(4 × 50 mL), combined organic layers were dried

over anhydrous Na SO , ﬁltered, and evaporated to give pure amine. M

3.2 g. Yield = 32%.

S-enantiomer (S) was synthesized by the same experimental pro-

cedures. Yield = 29%.

2

Cl

2

(2 × 50 mL). After that, solid K

2

CO

3

was added carefully (CO

2

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.77 (br. s., 2H), 3.01 (dd, J =

13.0, 7.9 Hz, 1H), 3.14 (dd, J = 13.0, 5.1 Hz, 1H), 4.54 (s, 2H),

5.18–5.27 (m, 1H), 5.31 (br. s., 1H), 6.73 (t, J = 3.7 Hz, 1H), 7.09 (d, J =

3.8 Hz, 1H), 7.29 (s, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.74 (d, J = 11.4 Hz,

1H), 8.17 (t, J = 7.9 Hz, 1H), 8.73 (d, J = 7.6 Hz, 1H), 11.95 (br. s., 1H).

2

4

=

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 45.7, 54.4, 59.8, 112.2 (d, J =

10.1 Hz), 112.9, 113.7 (dq, J = 26.7, 3.2 Hz), 114.1, 121.53 (qd, J = 7.4,

3.7 Hz), 123.5 (qd, J = 272.1, 2.8 Hz), 123.7 (d, J = 11.5 Hz), 127.1 (d,

J = 2.8 Hz), 128.1 (qd, J = 33.1, 8.3 Hz), 128.4, 128.6 (d, J = 3.7 Hz),

1

H NMR (CDCl

3

, 400 MHz): δ = 1.32 (s, 3H), 1.34 (s, 3H), 2.49 (br. s.,

3

5

H), 4.45–4.56 (m, 3H), 4.68 (s, 2H), 5.14–5.32 (m, 2H), 5.59 (s, 1H),

.89 (dddd, J = 16.8, 11.0, 5.7, 5.4 Hz, 1H), 7.10 (s, 1H).

157.6, 158.1 (d, J = 250.5 Hz), 160.3, 172.1.

1

3

ꢀ

C NMR (CDCl

3

, 100 MHz): δ = 23.5, 23.7, 56.0, 59.6, 60.5, 65.1,

HRMS (ESI) calcd for C18

H

15

F

4

N

4

O

2

S [Mꢀ H] 427.0857, found

1

14.5, 117.5, 132.9, 155.0, 156.3, 172.6.

427.0858.

4

.11.2. N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(4-

4

.9. General procedure D: For amide coupling

cyanophenyl)-1H-pyrrole-2-carboxamide

Compounds 3 (NBD-14260) and 4 (NBD-14261) were obtained

following the general procedure D and E from amine S17 and acid S4a.

Compounds were puriﬁed using column chromatography on silica gel

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

lowed by DMF (10 mL per 1 g of acid) and then HBTU (1 equiv.). The

resulting solution was stirred for 10 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 evapo-

3 3

(twice). Eluent 1: CHCl -MeOH (saturated with NH ~7M), 10:1 and 5:1,

Eluent 2: CH Cl -MeOH, 4:1 and 2:1.

2

3 (NBD-14260); (R): M = 207 mg. Yield = 17% (over two steps). rt =

+

rated, and the residue was dissolved in CH

2

Cl

2

(50 mL per 1 g of crude

1.077 min. Purity = 91%. LC-MS: m/z [M + H] = 368 Da.

product) and successively washed with 5% aqueous NaOH and 10%

tartaric acid solutions (25 mL per 1 g of crude product). The organic

4 (NBD-14261); (S): M = 288 mg. Yield = 21% (over two steps). rt =

+

1.217 min. Purity = 100%. LC-MS: m/z [M + H] = 368 Da.

1

layer was dried over anhydrous Na

2

4

SO , ﬁltered, evaporated, and dry

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.75 (br. s., 2H), 3.01 (dd, J =

loaded on silica. Eluting with hexane-EtOAc (1:1, then pure EtOAc) gave

the target compounds. Products were used in the next step without

analysis.

13.1, 7.8 Hz, 1H), 3.14 (dd, J = 13.2, 5.3 Hz, 1H), 4.54 (s, 2H),

5.17–5.26 (m, 1H), 5.31 (br. s., 1H), 6.84 (d, J = 3.9 Hz, 1H), 7.05 (d, J

= 3.9 Hz, 1H), 7.29 (d, J = 0.9 Hz, 1H), 7.81 (d, J = 8.0 Hz, 2H), 8.01 (d,

J = 8.3 Hz, 2H), 8.66 (d, J = 7.8 Hz, 1H), 12.04 (br. s., 1H).

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 45.7, 54.4, 59.8, 108.5, 109.6,

4

.10. General procedure E: For Allyl- and Alloc- deprotection

113.2, 114.1, 119.1, 125.1 (2C), 128.8, 132.7 (2C), 133.1, 136.1, 157.6,

1

60.4, 172.2.

ꢀ

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

HRMS (ESI) calcd for C18H N O S [M - H] 366.1030, found

16 5 2

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

PPh (10 mol%) was added under a nitrogen atmosphere followed by Pd

dba) (5 mol%). The mixture was stirred for 1 day under reﬂux. After

cooling, 50 mL of CH

with 10% aqueous K CO

366.1033.

3

(

2

4.11.3. N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(2,4-bis

(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

2

Cl

2

was added, and the organic phase was shaken

(50 mL) to remove the unreacted NDMBA. The

Compounds 5 (NBD-14313) and 6 (NBD-14314) were obtained

following the general procedure D and E from amine S17 and acid S4b.

Compounds were puriﬁed using column chromatography on silica gel

2

3

organic layer was separated, and the aqueous layer was extracted with

CH Cl

-EtOH (~4:1, (3–5) × 50 mL). Combined organic layers were

dried over anhydrous Na SO , ﬁltered, and concentrated. Puriﬁcation

by ﬂash chromatography (twice) (eluent 1: CHCl -MeOH (saturated

with NH ~7M), 10:1 and 5:1, eluent 2: CH Cl -MeOH, 4:1, 2:1, 1:1)

afforded amine as a slightly yellow or white solid.

2

(twice). Eluent 1: CHCl

3 3

-MeOH (saturated with NH ~7M), 10:1 and 5:1,

2

4

Eluent 2: CH Cl -MeOH, 4:1 and 2:1.

2

3

5 (NBD-14313); (S): M = 614 mg. Yield = 35% (over two steps). rt =

3

2

+

1.378 min. Purity = 100%. LC-MS: m/z [M + H] = 479 Da.

1

4

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

Scheme 5. Synthesis of compounds 1–38.

6

(NBD-14314); (R): M = 662 mg. Yield = 37% (over two steps). rt =

112.3 (d, J = 22.5 Hz), 112.7, 113.7 (qd, J = 32.4, 12.5 Hz), 113.8,

120.6 (d, J = 3.0 Hz), 122.9 (q, J = 271.5 Hz), 127.6 (q, J = 4.4 Hz),

129.6, 131.9 (d, J = 2.2 Hz), 138.5 (d, J = 9.0 Hz), 156.9, 159.4 (dq, J =

+

1

.365 min. Purity = 100%. LC-MS: m/z [M + H] = 479 Da.

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.89 (br. s., 2H), 3.00 (dd, J =

1

5

3.1, 7.9 Hz, 1H), 3.14 (dd, J = 13.2, 5.3 Hz, 1H), 4.54 (s, 2H),

.18–5.26 (m, 1H), 5.31 (br. s., 1H), 6.39 (d, J = 3.2 Hz, 1H), 7.06 (d, J

253.4, 2.2 Hz), 160.1, 176.4.

ꢀ

HRMS (ESI) calcd for C19

441.1014.

H

17

F

4

N O

2

S [M - H] 441.1014, found

=

3.9 Hz, 1H), 7.29 (s, 1H), 7.84 (d, J = 8.7 Hz, 1H), 8.07–8.13 (m, 2H),

8

.67 (d, J = 8.0 Hz, 1H), 12.04 (br. s., 1H).

13

C NMR (DMSO‑d

6

, 100 MHz): δ = 45.4, 54.0, 59.8, 111.6 (q, J =

4.11.5. N-(1-Amino-2-(4-(hydroxymethyl)thiazol-2-yl)propan-2-yl)-5-(4-

chloro-3,5-diﬂuorophenyl)-1H-pyrrole-2-carboxamide

3

2

.3 Hz), 112.2, 114.2, 123.3 (br.), 123.4 (q, J = 273.9 Hz), 123.5 (q, J =

72.4 Hz), 127.3 (q, J = 30.8 Hz), 128.1, 128.2 (q, J = 33.0 Hz), 128.9

Compound 8 (NBD-14280) was obtained following the general pro-

cedure D and Boc- and TBS-deprotection from amine racemic amine S25

and acid S28.

(

q, J = 3.0 Hz), 129.9, 133.7, 135.8, 157.7, 160.5, 172.1.

+

HRMS (ESI) calcd for C19

79.0971.

H

17

F

6

N

4

O

2

S [M + H] 479.0971, found

4

Deprotection: To a solution of protected thiazole from the previous

step in MeOH (10 mL), 1 M solution of HCl in MeOH (50 mL) was added

in one portion. The mixture was stirred for 3–4 h, and the solvent was

4

.11.4. N-(1-amino-2-(4-(hydroxymethyl)thiazol-2-yl)propan-2-yl)-5-(3-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

Compound 7 (NBD-14278) was obtained following the general pro-

cedure D and Boc- and TBS-deprotection from amine racemic amine S25

and acid S27.

evaporated. After that, 10% aqueous K

was extracted with CH Cl

-EtOH (~4:1, 4 × 50 mL). Combined organic

layers were dried over Na SO , ﬁltered, and concentrated. The com-

pound was puriﬁed using column chromatography on silica gel (twice).

Eluent 1: CHCl -MeOH (saturated with NH3~7M), 10:1 and 5:1, Eluent

2: CH Cl -MeOH, 5:1 and 2:1.

2 3

CO (50 mL) was added, and it

2

4

Deprotection: To a solution of protected thiazole from the previous

step in MeOH (10 mL), 1 M solution of HCl in MeOH (50 mL) was added

in one portion. The mixture was stirred for 3–4 h, and the solvent was

3

2

8 (NBD-14280): M = 471 mg. Yield = 35% (over two steps). rt =

+

evaporated. After that, 10% aqueous K

was extracted with CH Cl

-EtOH (~4:1, 4 × 50 mL). Combined organic

layers were dried over Na SO , ﬁltered, and concentrated. The com-

pound was puriﬁed using column chromatography on silica gel (twice).

Eluent 1: CHCl -MeOH (saturated with NH ~7M), 10:1 and 5:1, Eluent

-MeOH, 5:1 and 2:1.

2

CO

3

(50 mL) was added, and it

1.318 min. Purity = 100%. LC-MS: m/z [M + H] = 427 Da.

1

2

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.77 (s, 3H), 1.86 (br. s., 2H),

2

4

2.93 (d, J = 13.2 Hz, 1H), 3.12 (d, J = 13.2 Hz, 1H), 4.52 (s, 2H), 5.29

(br. s., 1H), 6.84 (d, J = 3.9 Hz, 1H), 6.99 (d, J = 3.9 Hz, 1H), 7.23 (s,

1H), 7.84 (d, J = 9.1 Hz, 2H), 8.12 (s, 1H), 11.93 (br. s., 1H).

3

1

3

2

1

: CH

2

Cl

2

C NMR (DMSO‑d

6

, 100 MHz): δ = 23.5, 51.7, 59.9, 60.3, 105.3 (t, J

7

(NBD-14278): M = 451 mg. Yield = 32% (over two steps). rt =

= 21.3 Hz), 108.3 (d, J = 23.2 Hz. 2C), 109.4, 112.5, 113.8, 129.1, 131.6

(t, J = 2.8 Hz), 132.8 (t, J = 10.3 Hz), 156.9, 158.3 (dd, J = 245.8, 4.4

Hz, 2C), 160.1, 176.4.

+

.350 min. Purity = 100%. LC-MS: m/z [M + H] = 443 Da.

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.77 (s, 3H), 1.82 (br. s., 2H),

2

7

.93 (d, J = 13.2 Hz, 1H), 3.12 (d, J = 13.1 Hz, 1H), 4.51 (d, J = 2.4 Hz,

H), 5.28 (br. s., 1H), 6.88 (d, J = 3.9 Hz, 1H), 7.01 (d, J = 3.9 Hz, 1H),

.23 (s, 1H), 7.73 (t, J = 8.1 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 8.00 (d, J

HRMS (ESI) calcd for C18

427.0811.

H

18ClF

2

4

N O

2

S [M + H]⁺427.0802, found

=

13.0 Hz, 1H), 8.15 (s, 1H), 12.04 (br. s., 1H).

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 23.5, 51.8, 59.9, 60.4, 109.9,

1

5

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

4

3

.11.6. N-(2-Amino-1-(4-(2-hydroxyethyl)thiazol-2-yl)ethyl)-5-(4-chloro-

,5-diﬂuorophenyl)-1H-pyrrole-2-carboxamide

J = 10.3 Hz), 112.9, 113.7 (dq, J = 26.4, 3.7 Hz), 114.1, 121.5 (qd, J =

7.2, 3.5 Hz), 123.5 (qd, J = 271.8, 2.2 Hz), 123.7 (d, J = 11.8 Hz), 127.1

(d, J = 2.2 Hz), 128.1 (qd, J = 33.2, 8.3 Hz), 128.4, 128.6 (d, J = 3.7 Hz),

Compounds 9 (NBD-14242) and 10 (NBD-14243) were obtained

following the general procedure D and E from amine S19 and acid S28.

154.0, 158.1 (d, J = 250.3 Hz), 160.3, 171.5.

ꢀ

Compounds were puriﬁed using column chromatography on silica gel

HRMS (ESI) calcd for C19

H

17

F

4

N O

2

S [M - H] 441.1015, found

(

twice). Eluent 1: CHCl

3

-MeOH (saturated with NH

3

~7M), 10:1 and 5:1,

441.1014.

Eluent 2: CH Cl -MeOH, 4:1 and 2:1.

2

9

(NBD-14242); (S): M = 292 mg. Yield = 22% (over two steps). rt =

4.11.9. N-(2-Amino-1-(4-(1,2-dihydroxyethyl)thiazol-2-yl)ethyl)-5-(3-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

+

1

.284 min. Purity = 95%. LC-MS: m/z [M + H] = 427 Da.

1

0 (NBD-14243); (R): M = 321 mg. Yield = 24% (over two steps). rt

Compounds 15 (NBD-14287) and 16 (NBD-14288) were obtained

following the general procedure D and E and F from amine S24 and acid

S27. Compounds were puriﬁed using column chromatography on silica

+

=

1.268 min. Purity = 96%. LC-MS: m/z [M + H] = 427 Da.

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.86 (br. s., 2H), 2.84 (t, J = 6.8

Hz, 2H), 3.00 (dd, J = 13.1, 7.9 Hz, 1H), 3.15 (dd, J = 13.1, 5.3 Hz, 1H),

3 3

gel (twice). Eluent 1: CHCl -MeOH (saturated with NH ~7M), 10:1, 5:1

3

2

.70 (t, J = 6.9 Hz, 2H), 4.68 (br. s., 1H), 5.17–5.26 (m, 1H), 6.85 (d, J =

.9 Hz, 1H), 7.04 (d, J = 3.9 Hz, 1H), 7.17 (s, 1H), 7.86 (d, J = 9.0 Hz,

H), 8.64 (d, J = 7.9 Hz, 1H), 11.96 (br. s., 1H).

and 3:1, Eluent 2: CH Cl -MeOH, 2:1 and 1:1.

2

Note that compounds 15 & 16 were obtained as a diastereomeric

mixture of 2 single compounds having the absolute conﬁguration of the

chiral carbon an as R for 16 and S for 15.

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 34.9, 45.8, 54.5, 60.2, 105.4 (t, J

=

21.5 Hz), 108.4 (d, J = 25.7 Hz, 2C), 109.5, 112.7, 114.1, 128.6, 131.9

15 (NBD-14287); (S): M = 365 mg. Yield = 19% (over two steps). rt

+

(

t, J = 2.8 Hz), 132.8 (t, J = 10.1 Hz), 154.0, 158.4 (dd, J = 245.9, 4.2

= 1.239 min. Purity = 96%. LC-MS: m/z [M + H] = 459 Da.

Hz, 2C), 160.4, 171.5.

16 (NBD-14288); (R): M = 402 mg. Yield = 21% (over two steps). rt

+

HRMS (ESI) calcd for C18

H16ClF

N

2 4

O

2

S [M - H]- 425.0656, found

= 1.304 min. Purity = 95%. LC-MS: m/z [M + H] = 459 Da.

1

4

25.0659.

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.80 (br. s., 2H), 3.01 (ddd, J =

1

3.2, 7.7, 1.7 Hz, 1H), 3.14 (dt, J = 13.2, 4.7 Hz, 1H), 3.44–3.52 (m,

4

.11.7. N-(2-Amino-1-(4-(2-hydroxyethyl)thiazol-2-yl)ethyl)-5-(3-ﬂuoro-

-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

1H), 3.71 (dt, J = 10.9, 3.9 Hz, 1H), 4.65 (dd, J = 6.7, 4.2 Hz, 1H), 4.71

(br. s., 1H), 5.17–5.26 (m, 1H), 5.35 (br. s., 1H), 6.89 (d, J = 3.9 Hz, 1H),

7.06 (d, J = 3.9 Hz, 1H), 7.30 (s, 1H), 7.74 (t, J = 8.1 Hz, 1H), 7.85 (d, J

= 8.4 Hz, 1H), 8.03 (d, J = 12.9 Hz, 1H), 8.68 (d, J = 7.8 Hz, 1H), 12.14

(br. s., 1H).

Compounds 11 (NBD-14258) and 12 (NBD-14259) were obtained

following the general procedure D and E from amine S19 and acid S27.

Compounds were puriﬁed using column chromatography on silica gel

(

twice). Eluent 1: CHCl

3

-MeOH (saturated with NH

3

~7M), 10:1 and 5:1,

¹³C NMR (DMSO‑d

6

, 100 MHz): δ = 45.7, (54.5, 54.5), (65.8, 65.8),

Eluent 2: CH Cl -MeOH, 4:1 and 2:1.

2

(71.3, 71.4), 110.0, 112.4 (d, J = 22.5 Hz), 112.9, 113.7 (qd, J = 32.6,

12.5 Hz), (114.3, 114.4), 120.6 (d, J = 3.0 Hz), 122.9 (q, J = 271.5 Hz),

127.6 (q, J = 3.0 Hz), 128.9, 132.0 (d, J = 1.8 Hz), 138.5 (d, J = 9.4 Hz),

(158.3, 158.4), 159.4 (dq, J = 251.2, 2.2 Hz), (160.3, 160.3), (171.7,

1

1 (NBD-14258); (R): M = 582 mg. Yield = 31% (over two steps). rt

+

=

1.333 min. Purity = 98%. LC-MS: m/z [M + H] = 443 Da.

1

2 (NBD-14259); (S): M = 506 mg. Yield = 27% (over two steps). rt

+

1.338 min. Purity = 96%. LC-MS: m/z [M + H] = 443 Da.

171.8).

1

ꢀ

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.79 (br. s., 2H), 2.84 (t, J = 6.8

HRMS (ESI) calcd for C19

457.0956.

H

17

F

4

N O

3

S [M - H] 457.0963, found

Hz, 2H), 3.00 (dd, J = 13.2, 7.9 Hz, 1H), 3.15 (dd, J = 13.2, 5.3 Hz, 1H),

3

1

.70 (t, J = 6.9 Hz, 2H), 4.67 (br. s., 1H), 5.18–5.26 (m, 1H), 6.89 (d, J =

.9 Hz, 1H), 7.06 (d, J = 3.9 Hz, 1H), 7.18 (s, 1H), 7.73 (t, J = 8.1 Hz,

H), 7.85 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 13.0 Hz, 1H), 8.67 (d, J = 7.9

4.11.10. N-(2-Amino-1-(4-(3-hydroxypropyl)thiazol-2-yl)ethyl)-5-(3-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

Hz, 1H), 12.06 (br. s., 1H).

Compounds 17 (NBD-14289) and 18 (NBD-142990) were obtained

following the general procedure D and E from amine S20 and acid S27.

Compounds were puriﬁed using column chromatography on silica gel

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 34.9, 45.8, 54.5, 60.3, 110.0,

1

2

12.4 (d, J = 22.5 Hz), 113.0, 113.3 (qd, J = 32.6, 12.7 Hz), 114.1,

20.6 (d, J = 2.6 Hz), 122.9 (q, J = 271.3 Hz), 127.6 (q, J = 3.1 Hz),

29.0, 132.1 (d, J = 1.8 Hz), 138.5 (d, J = 9.2 Hz), 154.1, 159.5 (dq, J =

3 3

(twice). Eluent 1: CHCl -MeOH (saturated with NH ~7M), 10:1 and 5:1,

Eluent 2: CH Cl -MeOH, 4:1 and 2:1.

2

51.3, 2.2 Hz), 160.4, 171.5.

17 (NBD-14289); (S): M = 519 mg. Yield = 27% (over two steps). rt

ꢀ

+

HRMS (ESI) calcd for C19

H

17

F

N

4 4

O

2

S [M - H] 441.1015, found

= 1.348 min. Purity = 95%. LC-MS: m/z [M + H] = 457 Da.

4

41.1015.

18 (NBD-14290); (R): M = 392 mg. Yield = 26% (over two steps). rt

+

=

1.370 min. Purity = 100%. LC-MS: m/z [M + H] = 457 Da.

1

4

.11.8. N-(2-Amino-1-(4-(2-hydroxyethyl)thiazol-2-yl)ethyl)-5-(2-ﬂuoro-

-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.66 (br. s., 2H), 1.74–1.83 (m,

2H), 2.71 (t, J = 7.6 Hz, 2H), 3.00 (dd, J = 13.2, 7.9 Hz, 1H), 3.15 (dd, J

= 13.1, 5.3 Hz, 1H), 3.44 (t, J = 6.4 Hz, 2H), 4.49 (br. s., 1H), 5.17–5.25

(m, 1H), 6.89 (d, J = 3.9 Hz, 1H), 7.06 (d, J = 3.9 Hz, 1H), 7.13 (s, 1H),

7.74 (t, J = 8.1 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 12.9 Hz,

1H), 8.67 (d, J = 7.9 Hz, 1H), 12.13 (br. s., 1H).

Compounds 13 (NBD-14273) and 14 (NBD-14274) were obtained

following the general procedure D and E from amine S19 and acid S4c.

Compounds were puriﬁed using column chromatography on silica gel

(

twice). Eluent 1: CHCl

3 3

-MeOH (saturated with NH ~7M), 10:1 and 5:1,

1

3

Eluent 2: CH Cl -MeOH, 4:1 and 2:1.

2

C NMR (DMSO‑d

6

, 100 MHz): δ = 27.6, 32.1, 45.8, 54.5, 60.2,

1

3 (NBD-14273); (S): M = 581 mg. Yield = 37% (over two steps). rt

110.0, 112.4 (d, J = 22.5 Hz), 112.9, 113.1, 113.7 (qd, J = 32.4, 12.5

Hz), 120.6 (d, J = 3.0 Hz), 122.9 (q, J = 271.3 Hz), 127.6 (q, J = 3.3 Hz),

129.0, 132.1 (d, J = 1.8 Hz), 138.5 (d, J = 9.4 Hz), 156.6, 159.4 (dq, J =

+

=

1.313 min. Purity = 100%. LC-MS: m/z [M + H] = 443 Da.

1

4 (NBD-14274); (R): M = 548 mg. Yield = 35% (over two steps). rt

+

1.308 min. Purity = 100%. LC-MS: m/z [M + H] = 443 Da.

251.2, 2.0 Hz), 160.4, 171.6.

1

ꢀ

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.80 (br. s., 2H), 2.84 (t, J = 6.9

HRMS (ESI) calcd for C20

455.1170.

H

19

F

4

N O

2

S [M - H] 455.1170, found

Hz, 2H), 3.00 (dd, J = 13.2, 7.9 Hz, 1H), 3.14 (dd, J = 13.1, 5.1 Hz, 1H),

3

1

.70 (t, J = 6.9 Hz, 2H), 4.68 (br. s., 1H), 5.18–5.26 (m, 1H), 6.73 (t, J =

.7 Hz, 1H), 7.09 (d, J = 3.9 Hz, 1H), 7.18 (s, 1H), 7.62 (d, J = 8.2 Hz,

H), 7.74 (d, J = 11.5 Hz, 1H), 8.17 (t, J = 8.0 Hz, 1H), 8.72 (d, J = 7.9

4.11.11. N-(2-Amino-1-(5-(1,2-dihydroxyethyl)thiazol-2-yl)ethyl)-5-(3-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

Hz, 1H), 12.01 (br. s., 1H).

Compounds 19 (NBD-14303) and 20 (NBD-14304) were obtained

following the general procedure D and E and F from amine S23 and acid

1

3

C NMR (DMSO‑d

6

, 100 MHz): δ = 34.8, 45.8, 54.5, 60.2, 112.2 (d,

1

6

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

S27. Compounds were puriﬁed using column chromatography on silica

25 (NBD-14267); (R): M = 471 mg. Yield = 35% (over two steps). rt

+

gel (twice). Eluent 1: CHCl

3

-MeOH (saturated with NH

3

~7M), 10:1, 5:1

= 1.289 min. Purity = 97%. LC-MS: m/z [M + H] = 442 Da.

and 3:1, Eluent 2: CH Cl -MeOH, 2:1 and 1:1.

2

26 (NBD-14268); (S): M = 493 mg. Yield = 36% (over two steps). rt

+

Note that compounds 19 & 20 were obtained as a diastereomeric

mixture of 2 single compounds having the absolute conﬁguration of the

chiral carbon an as R for 20 and S for 19.

= 1.285 min. Purity = 100%. LC-MS: m/z [M + H] = 442 Da.

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.99 (br. s., 2H), 3.06 (dd, J =

13.3, 7.5 Hz, 1H), 3.22 (dd, J = 13.2, 5.4 Hz, 1H), 5.20–5.30 (m, 1H),

6.90 (d, J = 3.9 Hz, 1H), 7.08 (d, J = 3.9 Hz, 1H), 7.59 (br. s., 1H),

7.70–7.78 (m, 2H), 7.86 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 13.0 Hz, 1H),

1

9 (NBD-14303); (S): M = 382 mg. Yield = 31% (over three steps). rt

+

=

1.316 min. Purity = 100%. LC-MS: m/z [M + H] = 459 Da.

2

0 (NBD-14304); (R): M = 216 mg. Yield = 27% (over three steps).

8.14 (s, 1H), 8.75 (d, J = 6.0 Hz, 1H), 11.95 (br. s., 1H).

+

13

rt = 1.230 min. Purity = 100%. LC-MS: m/z [M + H] = 459 Da.

C NMR (DMSO‑d

6

, 100 MHz): δ = 45.5, 54.5, 110.1, 112.5 (d, J =

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.68 (br. s., 2H), 3.00 (dd, J =

22.5 Hz), 113.2, 113.9 (qd, J = 32.4, 12.5 Hz), 120.7 (d, J = 2.8 Hz),

122.9 (q, J = 271.5 Hz), 124.0, 127.6 (q, J = 3.3 Hz), 128.8, 132.3 (d, J

= 2.0 Hz), 138.5 (d, J = 9.2 Hz), 150.1, 159.5 (dq, J = 251.4, 2.0 Hz),

1

3

5

3.2, 7.9 Hz, 1H), 3.14 (dd, J = 13.2, 5.3 Hz, 1H), 3.40–3.47 (m, 1H),

.51 (dd, J = 10.8, 6.2 Hz, 1H), 4.75 (t, J = 5.7 Hz, 1H), 4.94 (br. s., 1H),

.15–5.25 (m, 1H), 5.68 (br. s., 1H), 6.89 (d, J = 3.9 Hz, 1H), 7.06 (d, J

160.6, 162.5, 173.0.

ꢀ

=

3.9 Hz, 1H), 7.56 (s, 1H), 7.74 (t, J = 8.1 Hz, 1H), 7.85 (d, J = 8.3 Hz,

HRMS (ESI) calcd for C18

440.0811.

H

14

F

4

5

N O

2

S [M - H] 440.0810, found

1

H), 8.03 (d, J = 13.0 Hz, 1H), 8.66 (d, J = 7.9 Hz, 1H), 12.08 (br. s.,

H).

¹³C NMR (DMSO‑d

, 100 MHz): δ= (45.6, 45.7), 54.5, 66.7, 68.1,

4.11.15. N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(3-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-3-methyl-1H-pyrrole-2-carboxamide

Compounds 27 (NBD-14325) and 28 (NBD-14324) were obtained

following the general procedure D and E from amine S17 and acid S8.

Compounds were puriﬁed using column chromatography on silica gel

6

1

10.0, 112.4 (d, J = 22.6 Hz), 112.9, 113.7 (qd, J = 32.4, 12.5 Hz),

20.6 (d, J = 2.9 Hz), 122.9 (q, J = 271.3 Hz), 127.6 (q, J = 3.5 Hz),

29.0, 132.0 (d, J = 2.1 Hz), 138.5 (q, J = 2.3 Hz), 141.3, 141.4, 159.4

(

dq, J = 251.3, 1.9 Hz), 160.3, (171.2, 171.2).

+

HRMS (ESI) calcd for C19

59.1109.

H

19

F

4

N

4

O

3

S [M + H] 459.1109, found

3 3

(twice). Eluent 1: CHCl -MeOH (saturated with NH ~7M), 10:1 and 5:1,

4

Eluent 2: CH Cl -MeOH, 3:1 and 2:1.

2

7 (NBD-14325); (S): M = 454 mg. Yield = 27% (over two steps). rt

+

4

.11.12. 2-[2-Amino-1-[[5-[3-ﬂuoro-4-(triﬂuoromethyl)phenyl]-1H-

= 1.327 min. Purity = 100%. LC-MS: m/z [M + H] = 443 Da.

28 (NBD-14324); (R): M = 571 mg. Yield = 34% (over two steps). rt

pyrrole-2-carbonyl]amino]ethyl]thiazole-4-carboxylic acid

Compounds 21 (NBD-14271) and 22 (NBD-14272) were obtained

from direct NaOH hydrolysis of 100 mg of the corresponding methyl

esters 23 (NBD-14275) and 24 (NBD-14276).

+

= 1.336 min. Purity = 100%. LC-MS: m/z [M + H] = 443 Da.

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.81 (br. s., 2H), 2.32 (s, 3H),

3.04 (dd, J = 13.1, 7.2 Hz, 1H), 3.12 (dd, J = 13.1, 5.3 Hz, 1H), 4.55 (s,

2H), 5.20–5.27 (m, 1H), 5.31 (br. s., 1H), 6.74 (s, 1H), 7.31 (s, 1H),

7.72–7.82 (m, 2H), 7.87 (d, J = 12.7 Hz, 1H), 8.28 (d, J = 7.0 Hz, 1H),

11.60 (br. s., 1H).

2

1 (NBD-14271); (S): M = 51 mg. LC-MS: m/z [M + H]⁺= 443 Da.

+

2 (NBD-14272); (R): M = 60 mg. LC-MS: m/z [M + H] = 443 Da.

Due to broad NMR signals and since the corresponding crudes were

not active, no further characterization and puriﬁcation were performed.

¹³C NMR (DMSO‑d

, 100 MHz): δ = 12.9, 45.9, 54.0, 59.8, 112.0 (d,

6

J = 22.1 Hz), 112.3, 113.8 (qd, J = 32.4, 12.4 Hz), 114.2, 120.3 (d, J =

2.8 Hz), 122.9 (q, J = 271.5 Hz), 124.7, 125.8, 127.8 (q, J = 4.1 Hz),

129.9 (d, J = 1.8 Hz), 138.4 (d, J = 9.2 Hz), 157.7, 159.6 (dq, J = 251.6,

2.2 Hz), 160.8, 171.9.

4

1

.11.13. Methyl 2-(2-amino-1-(5-(3-ﬂuoro-4-(triﬂuoromethyl)phenyl)-

H-pyrrole-2-carboxamido)ethyl)thiazole-4-carboxylate

Compounds 23 (NBD-14275) and 24 (NBD-14276) were obtained

following the general procedure D and E from amine S15 and acid S27.

HRMS (ESI) calcd for C19

441.1013.

H

17

F

4

N

4

O

2

S [Mꢀ H]^ꢀ441.1014, found

Compounds were puriﬁed using column chromatography on silica gel

(

twice). Eluent 1: CHCl

3 3

-MeOH (saturated with NH ~7M), 10:1 and 5:1,

Eluent 2: CH Cl -MeOH, 4:1 and 2:1.

2

4.11.16. N-(2-Amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(3-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-3-methyl-1H-pyrrole-2-carboxamide

Compounds 29 (NBD-14329) and 30 (NBD-14328) were obtained

following the general procedure D and E from amine S18 and acid S8.

Compounds were puriﬁed using column chromatography on silica gel

2

3 (NBD-14275); (S): M = 344 mg. Yield = 21% (over two steps). rt

+

=

1.377 min. Purity = 100%. LC-MS: m/z [M + H] = 457 Da.

2

4 (NBD-14276); (R): M = 421 mg. Yield = 26% (over two steps). rt

+

1.394 min. Purity = 85%. LC-MS: m/z [M + H] = 457 Da.

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 2.04 (br. s., 2H), 3.06 (dd, J =

3 3

(twice). Eluent 1: CHCl -MeOH (saturated with NH ~7M), 10:1 and 5:1,

1

5

7

1

3.2, 7.7 Hz, 1H), 3.18 (dd, J = 13.2, 5.4 Hz, 1H), 3.82 (s, 3H),

.22–5.29 (m, 1H), 6.90 (d, J = 3.9 Hz, 1H), 7.07 (d, J = 3.9 Hz, 1H),

.74 (t, J = 8.1 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 12.9 Hz,

H), 8.44 (s, 1H), 8.82 (d, J = 6.4 Hz, 1H), 12.12 (br. s., 1H).

Eluent 2: CH Cl -MeOH, 3:1 and 2:1.

2

29 (NBD-14329); (S): M = 420 mg. Yield = 24% (over two steps). rt

+

= 1.326 min. Purity = 100%. LC-MS: m/z [M + H] = 443 Da.

30 (NBD-14328); (R): M = 620 mg. Yield = 35% (over two steps). rt

1

3

+

C NMR (DMSO‑d

6

, 100 MHz): δ = 45.4, 52.0, 54.5, 110.0, 112.4 (d,

= 1.318 min. Purity = 100%. LC-MS: m/z [M + H] = 443 Da.

1

J = 22.5 Hz), 113.1, 113.8 (qd, J = 32.4, 12.5 Hz), 120.7 (d, J = 2.8 Hz),

22.9 (q, J = 271.3 Hz), 127.6 (q, J = 3.3 Hz), 128.8, 129.1, 132.3 (d, J

H NMR (DMSO‑d

6

, 400 MHz): δ = 2.08 (br. s., 2H), 2.33 (s, 3H),

1

3.05 (dd, J = 13.1, 7.3 Hz, 1H), 3.14 (dd, J = 13.1, 5.1 Hz, 1H), 4.62 (s,

2H), 5.18–5.25 (m, 1H), 5.50 (br. s., 1H), 6.75 (s, 1H), 7.57 (s, 1H),

7.73–7.82 (m, 2H), 7.89 (d, J = 12.5 Hz, 1H), 8.32 (d, J = 6.8 Hz, 1H),

11.64 (br. s., 1H).

=

1.8 Hz), 138.5 (d, J = 9.2 Hz), 145.5, 159.5 (dq, J = 251.2, 2.2 Hz),

1

4

60.5, 161.3, 173.4.

HRMS (ESI) calcd for C19

55.0808.

ꢀ

15 4 4 3

H F N O S [M - H] 455.0806, found

¹³C NMR (DMSO‑d

, 100 MHz): δ = 12.9, 45.7, 54.0, 55.8, 112.0 (d,

6

J = 22.3 Hz), 112.3, 113.7 (qd, J = 32.6, 12.5 Hz), 120.3 (d, J = 2.8 Hz),

122.8 (q, J = 271.3 Hz), 124.6, 125.7, 127.8 (q, J = 3.5 Hz), 129.9 (d, J

= 2.0 Hz), 138.4 (d, J = 9.2 Hz), 139.0, 140.2, 159.5 (dq, J = 251.7, 2.4

Hz), 160.8, 171.5.

.11.14. 2-(2-Amino-1-(5-(3-ﬂuoro-4-(triﬂuoromethyl)phenyl)-1H-

pyrrole-2-carboxamido)ethyl)thiazole-4-carboxamide

Compounds 25 (NBD-14267) and 26 (NBD-14268) were obtained

following the general procedure D and E from amine S16 and acid S27.

Compounds were puriﬁed using column chromatography on silica gel

HRMS (ESI) calcd for C19

441.1014.

H

17

F

4

N

4

O

2

S [Mꢀ H]^ꢀ441.1014, found

(

twice). Eluent 1: CHCl

3 3

-MeOH (saturated with NH ~7M), 10:1 and 5:1,

Eluent 2: CH l2-MeOH, 3:1 and 1:1.

2

C

1

7

F. Curreli et al.

Bioorganic & Medicinal Chemistry 32 (2021) 116000

4

5

.11.17. N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)-2-methylpropyl)-

-(4-chloro-3,5-diﬂuorophenyl)-1H-pyrrole-2-carboxamide

437.1269.

Compounds 31 (NBD-14246) and 32 (NBD-14247) were obtained

4.11.20. N-(2-amino-1-(5-((2,3-dihydroxypropoxy)methyl)thiazol-2-yl)

ethyl)-5-(4-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

following the general procedure D and E from amine S21 and acid S28.

Compounds were puriﬁed using column chromatography on silica gel

Compounds 37 (NBD-14225) and 38 (NBD-14229) were obtained

following the general procedure D and E and F from amine S14 and acid

S26. Compounds were puriﬁed using column chromatography on silica

(

twice). Eluent 1: CHCl

3

-MeOH (saturated with NH3~7M), 10:1 and

-MeOH, 4:1 and 2:1.

1 (NBD-14246); (S): M = 364 mg. Yield = 28% (over two steps). rt

5

2 2

:1, Eluent 2: CH Cl

3

3 3

gel (twice). Eluent 1: CHCl -MeOH (saturated with NH ~7M), 10:1, 5:1

+

=

1.362 min. Purity = 95%. LC-MS: m/z [M + H] = 441 Da.

and 3:1, Eluent 2: CH Cl -MeOH, 2:1 and 1:1.

2

3

2 (NBD-14247); (R): M = 315 mg. Yield = 24% (over two steps). rt

Note that compounds 37 & 38 were obtained as a diastereomeric

mixture of 2 single compounds having the absolute conﬁguration of the

chiral carbon as R for 38 and S for 37.

+

1.377 min. Purity = 96%. LC-MS: m/z [M + H] = 441 Da.

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.08 (s, 3H), 1.13 (s, 3H), 1.97

(

br. s., 2H), 4.57 (s, 2H), 5.29 (d, J = 7.6 Hz, 1H), 5.33 (br. s., 1H), 6.84

d, J = 3.9 Hz, 1H), 7.08 (d, J = 3.9 Hz, 1H), 7.32 (s, 1H), 7.83 (d, J =

37 (NBD-14225); (S): M = 342 mg. Yield = 17% (over two steps). rt

+

= 1.342 min. Purity = 100%. LC-MS: m/z [M + H] = 485 Da.

8

.9 Hz, 2H), 8.26 (d, J = 8.4 Hz, 1H), 12.02 (br. s., 1H).

38 (NBD-14229); (R): M = 381 mg. Yield = 19% (over two steps). rt

1

3

+

C NMR (DMSO‑d

6

, 100 MHz): δ = 27.6, 28.5, 52.7, 59.2, 59.8,

= 1.456 min. Purity = 95%. LC-MS: m/z [M + H] = 485 Da.

1

2

05.4 (t, J = 21.3 Hz), 108.4 (d, J = 25.4 Hz, 2C), 109.5, 113.2, 114.4,

28.4, 132.0 (t, J = 2.8 Hz), 132.8 (t, J = 10.1 Hz), 157.3, 158.4 (dd, J =

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.77 (br. s., 2H), 3.04 (dd, J =

13.1, 7.8 Hz, 1H), 3.17 (dd, J = 13.3, 5.4 Hz, 1H), 3.29–3.36 (m, 3H),

3.45 (dd, J = 9.9, 4.6 Hz, 1H), 3.54–3.61 (m, 1H), 4.65 (s, 2H), 4.72 (br.

s., 2H), 5.19–5.28 (m, 1H), 6.79 (d, J = 3.9 Hz, 1H), 7.06 (d, J = 3.8 Hz,

1H), 7.66 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.2 Hz, 2H), 8.67

46.4, 4.1 Hz, 2C), 160.0, 170.2.

ꢀ

HRMS (ESI) calcd for C19

39.0813.

H18ClF

N

2 4

O

2

S [M - H] 439.0813, found

4

(

d, J = 7.8 Hz, 1H), 12.00 (br. s., 1H).

13

4

5

.11.18. N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)-2-methylpropyl)-

-(4-(triﬂuoromethyl)phenyl)-1H-pyrrole-2-carboxamide

C NMR (DMSO‑d

6

, 100 MHz): δ = 45.6, 54.6, 63.0, 64.5, 70.5,

71.8, 108.9, 113.1, 124.4 (q, J = 271.6 Hz), 125.1 (2C), 125.6 (q, J =

3.7 Hz, 2C), 126.6 (q, J = 32.2 Hz), 128.3, 133.3, 135.4, 135.6, 141.0,

160.5, 173.1.

Compounds 33 (NBD-14248) and 34 (NBD-14249) were obtained

following the general procedure D and E from amine S21 and acid S26.

Compounds were puriﬁed using column chromatography on silica gel

HRMS (ESI): Calcd for C21

H

24

F

N

3 4

O

4

S [M + H]⁺485.1465, found

(

twice). Eluent 1: CHCl

3

-MeOH (saturated with NH3~7M), 10:1 and

-MeOH, 4:1 and 2:1.

3 (NBD-14248); (S): M = 435 mg. Yield = 27% (over two steps). rt

485.1466.

5

2 2

:1, Eluent 2: CH Cl

3

+

Declaration of Competing Interest

=

1.354 min. Purity = 97%. LC-MS: m/z [M + H] = 439 Da.

3

4 (NBD-14249); (R): M = 501 mg. Yield = 31% (over two steps). rt

+

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

1.360 min. Purity = 99%. LC-MS: m/z [M + H] = 439 Da.

1

H NMR (DMSO‑d

6

, 400 MHz): δ = 1.09 (s, 3H), 1.14 (s, 3H), 1.95

(

br. s., 2H), 4.58 (s, 2H), 5.30 (d, J = 8.2 Hz, 1H), 5.38 (br. s., 1H), 6.78

d, J = 3.8 Hz, 1H), 7.08 (d, J = 3.8 Hz, 1H), 7.32 (s, 1H), 7.71 (d, J =

8

.4 Hz, 2H), 8.02 (d, J = 8.2 Hz, 2H), 8.28 (d, J = 8.8 Hz, 1H), 12.05 (br.

Acknowledgments

s., 1H).

¹³C NMR (DMSO‑d

6

, 100 MHz): δ = 27.6, 28.5, 52.7, 59.2, 59.8,

This study was supported by funds from NIH Grant R01 AI104416

(AKD), and the New York Blood Center (AKD).

1

4

1

08.8, 113.5, 114.4, 124.4 (q, J = 272.1 Hz), 125.1 (2C), 125.6 (q, J =

.1 Hz, 2C), 126.7 (q, J = 31.7 Hz), 128.2, 133.4, 135.6, 157.3, 160.0,

70.2.

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N

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2

S [M - H] 437.1265, found

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37.1269.

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Article Doi

Design, synthesis, and antiviral activity of a series of CD4-mimetic small-molecule HIV-1 entry inhibitors

DOI: 10.1016/j.bmc.2021.116000

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Full text of DOI:10.1016/j.bmc.2021.116000

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