Structural Optimization of 2,3-Dihydro-1H-cyclopenta[ b]quinolines Targeting the Noncatalytic RVxF Site of Protein Phosphatase 1 for HIV-1 Inhibition

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Structural Optimization of 2,3-Dihydro-1H-cyclopenta[b]quinolines

Targeting the Noncatalytic RVxF Site of Protein Phosphatase 1 for

HIV ‑1 Inhibition

Xionghao Lin, Ayyiliath M Sajith, Songping Wang, Namita Kumari, Meng S Choy, Asrar Ahmad,

Dana R. Cadet, Xinbin Gu, Andrey I. Ivanov, Wolfgang Peti, Amol Kulkarni,* and Sergei Nekhai*

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sı Supporting Information

ABSTRACT: Combination antiretroviral therapy (cART) suppresses

human immunodeﬁciency virus-1 (HIV-1) replication but is unable to

permanently eradicate HIV-1. Importantly, cART does not target HIV-1

transcription, which is reactivated in latently infected reservoirs, leading to

HIV-1 pathogenesis including non-infectious lung, cardiovascular, kidney,

and neurodegenerative diseases. To address the limitations of cART and to

prevent HIV-1-related pathogenesis, we developed small molecules to target

the noncatalytic RVxF-accommodating site of protein phosphatase-1 (PP1)

to prevent HIV-1 transcription activation. The PP1 RVxF-accommodating

site is critical for the recruitment of regulatory and substrate proteins to PP1. Here, we conﬁrm that our previously developed 1E7-

0

1

3 compound binds to the PP1 RVxF-accommodating site. Iterative chemical alterations to 1E7-03 furnished a new analogue, HU-

a, with enhanced HIV-1 inhibitory activity and improved metabolic stability compared to 1E7-03. In a Split NanoBit competition

assay, HU-1a primarily bound to the PP1 RVxF-accommodating site. In conclusion, our study identiﬁed HU-1a as a promising HIV-

transcription inhibitor and showed that the PP1 RVxF-accommodating site is a potential drug target for the development of novel

1

HIV-1 transcription inhibitors.

KEYWORDS: HIV-1 transcription, protein phosphatase-1, RVxF-accommodating site, structural optimization

uman immunodeﬁciency virus-1 (HIV-1) has emerged

as a pathogen with signiﬁcant worldwide burden.

Approximately 37.9 million people are living with HIV-1 and

there are ∼1.7 million annual new infections. A major barrier

for an HIV-1/AIDS cure is the ability of HIV-1 to resist both

to identify novel targets or drugs, which can prevent HIV-1

1

H

transcription activation, to address the limitations of cART and

reduce HIV-1-related lung, cardiovascular, kidney, and neuro-

logical disorders.

Protein phosphatase-1 (PP1) belongs to the protein

phosphatase (PPP) superfamily. The substrate speciﬁcity of

PP1 dimer depends on its regulatory protein subunit that

targets a catalytic subunit (PP1α, PP1β, or PP1γ) into a

immune surveillance and combination antiretroviral therapy

2

(

cART). cART eﬃciently suppresses HIV-1 replication but is

3

unable to eradicate the virus and cure infected patients.

Moreover, HIV-infected individuals continue to be at elevated

risk of developing chronic non-infectious lung, cariovascular,

11

speciﬁc cellular location. Over 200 validated regulatory

proteins bind to PP1 catalytic subunits via multiple docking

motifs, including RVxF, SpiDoc, SILK, MyPhoNE, ΦΦ, and

4

kidney, and neurodegenerative diseases despite cART. HIV-1

12,13

reservoirs that harbor latent integrated provirus can produce

viral proteins and also become reactivated when cART is

NIPP1-helix.

Our previous studies showed that PP1 is

involved in HIV-1 transcription by binding Tat Val-36 and

Phe-38 residues within Tat QVCF sequence to the PP1 RVxF-

5

−7

interrupted or drug resistance appears.

Transcription

activation is an essential step in the reactivation of latent

HIV-1. Although numerous HIV transcription inhibitors have

been discovered to target various host and viral proteins and

RNA, including HIV-1 Tat, transactivation response element

14

accommodating site. On the basis of this ﬁnding, we

designed small molecules to target the RVxF-accommodating

site of PP1, to disrupt the interaction between PP1 and HIV-1

(

TAR) RNA, positive transcription elongation factor b (P-

Received: July 16, 2020

TEFb), or their respective interactions, none of them are

clinically available because of their toxicity or the absence of in

8

vivo anti-HIV activity. A recently identiﬁed promising HIV-1

Tat inhibitor, Dideydro-Cortistatin A (dCA), delays viral

9

,10

rebound in mouse model after cART interruption

but has

not yet been tested in a clinical trial. Therefore, it is imperative

©

XXXX American Chemical Society

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Figure 1. Identiﬁcation of PP1-binding site for 1E7-03 by protein painting. (A) Heat map of peptides derived from PP1 and quantiﬁed in the

following groups: (i) unpainted PP1, (ii) PP1 painted by molecule ELNLLB, and (iii) PP1 pretreated with 1E7-03 and then painted by ELNLLB.

The scale from 0−1 represents the ratio of group ii or iii vs group i. (B) Visualization of 1E7-03 binding sites on PP1. All cleavage sites except Lys-

111 and Arg-122 were mapped on the surface of PP1 (blue for the sites not aﬀected by 1E7-03 binding and red for sites protected by 1E7-03). The

model of PP1 was generated with PyMOL 1.5. (C) SIEVE quantitative data for the selected peptides that were protected by 1E7-03 from ELNLLB

painting. Ion elution proﬁles (top) and integrated intensities (bottom) are shown in blue for group i, in red for group ii, and in green for group iii.

Data are shown in triplicate, and the ratios between the three groups are indicated.

Tat and inhibit HIV-1 transcription. Compound 1H4 was

identiﬁed as the initial lead with an IC ∼ 10 μM and CC =

that 1E7-03 reduced IL-6 levels, improved bleeding score,

decreased lung injury, and prevented death of HIV-1

transgenic mice challenged with LPS. We attributed this

5

0

50

1

5

18

1

00 μM. Given the moderate activity and relatively narrow

therapeutic window, we performed structural optimization of

eﬀect of 1E7-03 to the inhibition of HIV-1 gene expression in

macrophages which restored macrophage migration into the

lungs and decreased lung neutrophil inﬁltration in HIV-1

1

H4 using structure−activity relationship (SAR). Initial SAR

studies led to the identiﬁcation of compound 7 as the new lead

compound with an IC ∼ 0.9−4 μM and CC ∼ 10−16

μM. Both compounds, 1H4 and 7, have been shown to

18,19

transgenic mice.

While 1E7-03 had potent eﬀects in vivo,

50

1

6

its metabolic stability was poor because of its facile conversion

17

prevent the interaction of PP1 with Tat as well as the

into degradation products 1 (DP1) and DP3. Both of the

degradation products eﬃciently bound PP1 in vitro but lacked

cell-permeability and, thus, did not display HIV-1 inhibitory

15,16

translocation of PP1 into nucleus.

Because of the high

cytotoxicity of compound 7, we further optimized it and

identiﬁed compound 1E7-03 that was less toxic (CC₅₀∼100

μM) and showed potent HIV-1 inhibitory activity (IC₅₀∼ 4.5

μM). 1E7-03 also prevents translocation of PP1 by Tat into

17

activity. Therefore, the poor metabolic stability of 1E7-03 is

likely to impair its future application as antiviral therapeutics.

In this study, we demonstrate that 1E7-03 binds to the

RVxF-accommodating site of PP1 using the “protein painting”

approach that was combined with molecular docking and Split

NanoBit competition assay. We further optimized the structure

1

6

the nucleus. We showed antiviral eﬃciency of 1E7-03 in

HIV-1 infected humanized mice in which it reduced HIV-1

17

mRNA production by ∼40-fold. We also recently showed

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Figure 2. Molecular docking of 1E7-03 binding to PP1. (A) PP1 model showing docking of 1E7-03. In total, 295 binding positions were generated

using docking tool described in Methods. These binding sites were sorted into 13 clusters according to the binding energy (from −9.3 to −6.7

kcal/mol). The clusters are shown on the PP1 molecule generated by PyMOL 1.5. (B) 1E7-03 binding to the RVxF-accommodating site of PP1

(C-8, −7.2 kcal/mol) is shown. 1E7-03 and its hydrogen bonding residues are shown as sticks. Indicated green dashes are hydrogen bonds formed

with residues Asp-242, Leu-289, and Phe-293. (C) 1E7-03 binding to the MYPT (myosin phosphatase target) site of PP1 is shown. 1E7-03 and its

hydrogen bonding residues are shown as sticks. Indicated green dashes are hydrogen bonds formed with Trp-216 and His-237 (−7.6 kcal/mol,

position C-3). (D) 1E7-03 binding to the NIPP1helix-PP1 interface is shown. 1E7-03 and its hydrogen bonding residues are shown as sticks.

Indicated green dashes are hydrogen bonds formed with residues Arg-43, Ile-51, and Arg-191 (C-12, −6.8 kcal/mol).

of 1E7-03 by synthesizing 49 analogues that had modiﬁcations

at the chemically labile sites of 1E7-03 with the introduction of

more stable substructures such as urea and piperazine motifs.

The HIV-1 inhibitory activity of 1E7-03 analogues was ﬁrst

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Figure 3. Synthesis of 1E7-03 analogues. (A) Previously conducted structural analysis of 1E7-03 indicated labile sites and essential

pharmacophores for its HIV-1 inhibitory activity. (B) Synthetic scheme of 1E7-03 analogue preparation. DMF, dimethylformamide; TEA,

triethylamine; DCM, dichloromethane; T P, propylphosphonic anhydride.

3

evaluated in one round HIV-1 infection using VSV-G

pseudotyped HIV-1 expressing luciferase (HIV-1-LUC-G).

The activity was also tested using fully replication competent

T-tropic HIV-1 IIIB. The metabolic stability of the best

analogue HU-1a was analyzed in human or mouse plasma and

liver microsomes using liquid chromatography Fourier trans-

form mass spectrometry (LC-FT/MS) analysis. PP1 binding of

HU-1a was tested using the Split NanoBit approach along with

molecular docking.

RESULTS AND DISCUSSION

^■1

E7-03 Binds to the PP1 RVxF Site. Using surface

plasmon resonance (SPR), we previously showed that the

HIV-1 inhibitory compound 1E7-03 binds to PP1 and

17

competes with the QVCF motif-containing pRb-Tat peptide.

A 1E7-03 analogue, C31, which eﬃciently inhibits Ebola virus

2

0,21

(EBOV) replication,

primarily binds to the C-terminal

21

groove of PP1. Recently, we used a “protein painting”

technique to test PP1 binding of the EBOV inhibitory

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Table 1. Analogues of 1E7-03

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Table 1. continued

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Table 1. continued

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0,21

compound, 1E7-07.

Using this technique in combination

on their binding energy (range from −9.3 to −6.7 kcal/mol)

and binding conﬁguration (Figure 2A). 1E7-03 was docked

into the RVxF-accommodating site of PP1 with an energy of

−7.2 kcal/mol (C-8 site, Figure 2A), and its N-[(ethylamino)-

carbonyl]acetamide chain formed hydrogen bonds with

residues Asp-242 and Leu-289 (Figure 2B). Also, the hydrogen

bonding between the methoxy group and Phe-293 residue

further enhanced the binding of 1E7-03 with PP1 (Figure 2B).

Mutation of the cdNIPP1 RVxF motif eliminated the eﬀect of

with molecular docking and Split NanoBiT assay, we showed

that 1E7-07 bound to the PP1 RVxF site. We utilized this

protein painting” technique to map the 1E7-03 binding sites

20

“

onto PP1. Brieﬂy, we employed a small molecule “paint”, EZ-

Link NHS-LC-LC-Biotin (ELNLLB), which covalently

modiﬁes surface lysine and arginine residues of PP1. Thus,

only non-painted lysine and arginine PP1 residues were

accessible for trypsin digestion. Trypic peptides were detected

using LC-FT/MS analysis (Figure 1A, group i). Addition of

the ELNLLB “paint” protected surface-exposed lysine and

arginine residues and blocked trypsin digestion, resulting in

20

1E7-03 on the PP1:cdNIPP1 interaction, suggesting that

1E7-03 primarily binds to the PP1 RVxF-accommodating

groove and protects Lys-168 from ELNLLB painting (Figure

1B). Docking also predicted 1E7-03 binding to the C-2 pocket

with binding energy −9.1 kcal/mol (Figure 2A), which might

protect the Arg-221 residue observed in the painting analysis

(Figure 1B). However, the C-2 pocket is located in close

proximity to the catalytic site of PP1, and 1E7-03 had no eﬀect

“

painted” PP1 (Figure 1A, group ii). Pretreatment with the

inhibitor 1E7-03 led to competitive exclusion of ELNLLB

paint within the compound binding site and unmasking of the

trypsin cleavage sites (Figure 1A, group iii). Twenty-nine PP1-

derived peptides that represented 74.6% of PP1 sequence were

quantiﬁed label-free by LC-FT/MS followed by SIEVE 2.1

analysis. Compared to the non-painted PP1, ELNLLB painting

reduced the intensities of peptide signals by more than 80%

Figure 1A, compare groups i and ii). All trypsin cleavage sites

were located on the surface of PP1 with the exception of Lys-

11 and Arg-122. Experimentally accessible sites are shown in

Figure 1B, with red color indicating 1E7-03 protected sites and

blue color indicating all paint-accessible sites that were not

protected. Pretreatment with 1E7-03 protected several

residues including Lys-168, Arg-187, Arg-188, Arg-221, and

Lys-238 (Figure 1B, labeled with red color). These residues are

20

on PP1 enzymatic activity. Therefore, 1E7-03 is not likely to

bind to this site. In addition, docking data indicated that 1E7-

03 could bind to the MYPT1 (myosin phosphatase target)

11

(

pocket of PP1 (C-3, binding energy −7.6 kcal/mol), where it

formed hydrogen bonds with residues Trp-216 and His-237

(Figure 2C), thus protecting Lys-238 from being painted.

Contrary to the previous SAR data, the N-[(ethylamino)-

carbonyl]acetamide chain of 1E7-03 was found to be non-

1

16,17

essential for interaction with PP1.

These results indicate

that 1E7-03 is not likely to bind to the C-3 pocket of PP1.

1E7-03 was also docked to the C-terminal groove of PP1

(Figure 2A, C-1, −9.3 kcal/mol) and NIPP1 helix-binding site

(NIPP1 helix−PP1 interface) (Figure 2A, C-12, −6.8 kcal/

mol)). In the latter, 1E7-03 formed hydrogen bonds with

residues Arg-43, Ile-51, and Arg-191 (Figure 2D), protecting

Arg-187 and Arg-188 from ELNLLB painting (Figure 1B).

However, the mutation of PP1 within the C-terminal groove

and also within the cdNIPP1 helix motif only slightly aﬀected

2

39

246 169

located within peptides HDLDLIcR

,

IFccHGGLSPDL-

1

87

189

QSmEQIR , and

ImRPTDVPDQGLLcDLLWSDPDKD-

2

21

VQGWGENDR (Figure 1C). 1E7-03 protection led to the

restoration of the peptides ion current intensity signals to over

0% of the control group i (Figure 1C). In contrast, peptides

7

with the sites that were not protected showed little or no

change in ion current intensities (Figure S1). Taken together,

these data suggest that 1E7-03 interaction with PP1 protects

residues Lys-168, Arg-187, Arg-188, Arg-221, and Lys-238.

To further deﬁne the 1E7-03 binding site on PP1, we

utilized our previous analysis of the eﬀect of 1E7-03 on the

interaction of PP1:cdNIPP1 and their mutants using the Split

NanoBit system which showed that 1E7-03 interacted

primarily with the RVxF site. We combined these previous

data with the molecular docking of 1E7-03 on PP1 (PDB:

E7A). Using molecular docking, 295 binding conﬁgurations

were generated and grouped into 13 clusters (C1−C13) based

20

the competition of 1E7-03 on PP1:cdNIPP1 interaction,

suggesting that 1E7-03 can only weakly interact with either the

PP1 C-terminal groove or the cdNIPP1 helix pocket. To

summarize our protein painting, molecular docking, and

previous Split NanoBit results, 1E7-03 binds to the RVxF-

accommodating site, the C-terminal groove, and NIPP1 helix

pocket of PP1 with the RVxF site being the major binding site.

Synthesis of 1E7-03 Analogues. In previous studies, we

identiﬁed the amide and ester bonds in the linear linker as the

20

3

17

major labile sites of 1E7-03 (Figure 3A). We also found that

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Figure 4. Inhibition of one round of HIV-1 infection by 1E7-03 analogues. (A) High throughput screening of HIV-1 inhibitory 1E7-03 analogues.

CEM T cells were infected with HIV-1-LUC-G and treated with the indicated compounds for 24 h. HIV-1-expressed luciferase activity is shown as

percent of control treated with DMSO vehicle. Data are the means of triplicates. (B) Cell viability screening of 1E7-03 analogues using MTT assay.

CEM T cells were treated with indicated compounds for 24 h. Cell viability is shown as percent of control treated with DMSO vehicle. Data are the

means of triplicates. (C) Index of HIV-1 inhibition or activation by 1E7-03 analogues at 10 μM concentration. The index was calculated by the

formula 1 − (percent of cell viability/percent of HIV-1 replication). Red indicates HIV-1 inhibition, and blue indicates HIV-1 activation. (D) Dose-

dependent HIV-1 inhibition by 1E7-03, HU-1a, HU-1c, and HU-1d. CEM T cells were infected with HIV-1-LUC-G and treated with serially

diluted 1E7-03 or the analogues. HIV-1 inhibition was plotted as percent of DMSO-treated control. Data are the mean ± SD of triplicates. (E)

Cytotoxicity of 1E7-03, HU-1a, HU-1c, and HU-1d. CEM T cells were treated with serially diluted compounds for 24 h. The cell viability was

analyzed by MTT assay. Data are the mean ± SD of triplicates.

HIV-1 inhibitory activity and toxicity of 1E7-03 analogues

were aﬀected by electrostatic interactions of R and R

aimed at improving the metabolic stability and HIV-1

inhibitory activity of 1E7-03 by replacing the amide and

ester groups in the 1E7-03 side chain that were previously

1

2

15,16

moieties (Figure 3A).

Therefore, the current study was

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Figure 5. Inhibition of HIV-1 IIIB infection and metabolic stability of 1E7-03 analogues. (A and B) HIV-1 gag mRNA was analyzed in HIV-1 IIIB

infected CEM T cells and HIV-1 IIIB infected PBMCs (see Methods for details). RNA was extracted from the infected cells treated with DMSO,

10 μM 1E7-03, 10 μM HU-1a, 10 μM HU-1c, 10 μM HU-1d, or 25 μM AZT for 72 h. RNA expression was analyzed by RT-PCR using 18S

mRNA for normalization. The means ± SD are shown (n = 3 for all panels). * p < 0.05; ** p < 0.01; compound treated groups vs untreated control

or HU-1a vs 1E7-03. (C) Analysis of p24 in media collected from HIV-1 IIIB infected CEM T cells. Cells were infected with HIV-1 IIIB and

treated with compounds as described above. Culture supernatants were collected, and p24 levels were quantiﬁed by ELISA. Data are means ± SD

of triplicates. * p < 0.05; ** p < 0.01; compound treated groups vs untreated control or HU-1a vs. 1E7-03. (D) Expression of PP1 prevents HIV-1

transcription inhibition. 293T cells were transfected with plasmids expressing HIV-1 LTR luciferase and Tat, or HIV-1 LTR luciferase, Tat, and

PP1 and treated with 10 μM compounds as described in Methods. Luciferase activity is shown as means ± SD of triplicates. * p < 0.01, control vs

PP1 expressing samples for HU-1a and 1E7-03. (E) Stability of HU-1a and 1E7-03 in human and mouse plasma. HU-1a or 1E7-03 (10 μM) were

added to human or mouse plasma and incubated at 37 °C for 6 h. Samples were collected at the diﬀerent time points, and compounds were

extracted and quantiﬁed by LC/FT-MS analysis. Data are means ± SD of triplicates. (F) Stability of HU-1a and 1E7-03 in liver microsomes from

human and mouse. HU-1a or 1E7-03 were mixed with liver microsomes to the ﬁnal concentration of 10 μM and incubated at 37 °C for 24 h.

Samples were collected at the diﬀerent time points, and compounds were extracted and analyzed by LC/FT-MS. Data are means ± SD of

triplicates. * p < 0.05; ** p < 0.01, and *** p < 0.001.

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identiﬁed as hydrolytically-labile (Figure 3A). We also studied

the impact of the substitutions in the arylidene and the side-

chain moieties (R₁and R₂respectively) on the HIV-1

inhibitory activity and overall stability of 1E7-03. We

envisioned that changing the ester moiety into an amide and

altering the electrostatics of amide moiety will furnish

compounds with superior stability. As indicated in the scheme

shown in Figure 3B, target compounds were synthesized from

the commercially available isatin (1), which was subjected to a

Pﬁtzinger reaction with cyclopentanone to form 2,3-dihydro-

signiﬁcance of R₁group. Next, cell viability of 1E7-03

analogues was measured using MTT approach (Figure 4B),

and index of HIV-1 inhibition or activation by analogues at 10

μM was calculated according to the formula 1 − (% of HIV-1

replication /% of cell viability) (Figure 4C and Table 1). As

shown in Figure 4B,C, and Table 1, top HIV-1 inhibitory

compounds HU-1a, HU-1c, and HU-1d showed no or

moderate cellular toxicity and an inhibitory index. Of note, a

number of compounds including HU-2e−HU-2h showed

considerable indexes of HIV-1 inhibition (Figure 4C),

apparently because they promoted CEM T cell growth (Figure

4B). Thus, compounds HU-1a, HU-1c, and HU-1d were

chosen as top candidates for further analysis. Next, we

compared these compounds with 1E7-03 for HIV-1 inhibition

and cytotoxicity. HIV-1 inhibition was accessed in HIV-1-

LUC-G infected CEM T cells treated with the indicated

concentrations of each compound for 24 h as described above.

Cytotoxicity was also evaluated by MTT assay in CEM T cells

treated with the indicated concentrations of each compound

for 24 h. Compounds HU-1a and HU-1d inhibited one round

of HIV-1 replication with slightly lower IC₅₀(IC₅₀= 3.7 μM

and IC₅₀= 4.5 μM) than 1E7-03 (IC₅₀= 4.8 μM) whereas

HU-1d had slightly higher IC₅₀= 7.5 μM (Figure 4D).

Cytotoxicity analysis showed that HU-1c, HU-1d, and 1E7-03

exhibited approximately 50% cytotoxicity at 100 μM, whereas

HU-1a had less than 25% cytotoxicity at 100 μM (Figure 4E).

Therefore, HU-1a appeared to be the most advanced

candidate as it showed the strongest HIV-1 inhibitory activity

and the lowest cytotoxicity.

1

H-cyclopenta[b]quinoline-9-carboxylic acid (2). Knovenege-

type condensation between 2 and substituted benzaldehydes

generated carboxylic acids 3 and 4. Coupling with diﬀerentially

substituted 1° and 2° amines generated the desired HU

analogues. The modular synthetic route was amenable to the

structural variations in R and R groups and was ideally suited

1

2

for the generation of the SAR library. We converted the 1°-

amine in HU-5 into urea (HU-1a to HU-1k), secondary amide

(HU-2a to HU-2j), and sulfonamide (HU-3a and HU-3b) to

evaluate the eﬀect of nitrogen substituents on the overall

stability of the resultant analogues. We hypothesized that

addition of a sterically bulky tertiary amide may render the

analogues more resistant to hydrolysis. To test this hypothesis,

we developed piperazine analogues (HU-7a to HU-7d).

Analogously, we developed analogues (HU-8a to HU-8e)

using substrate 4. Finally, analogues HU-9a−HU-9f were

synthesized to study the signiﬁcance of the arylidene moiety on

the biological activity (Figure 3B and Table 1).

Inhibition of One Round HIV-1 Infection with VSV-G

Pseudotyped Virus. To evaluate the HIV-1 inhibitory

activity of synthesized analogues, CEM T cells were infected

with VSV-G pseudotyped pNL4-3.Luc.R-E-virus (HIV-1-LUC-

G) for 24 h and then treated with 3, 10, and 30 μM of each

compound for another 24 h. Cells were lysed, and luciferase

activity was measured to assess HIV-1 replication (Figure 4A).

Compounds HU-1a, HU-1c, HU-1d, HU-2c, HU-7c, and

HU-8e inhibited HIV-1 by ≥75% at 30 μM concentration

compared with the DMSO control (Figure 4A). Moreover,

compounds HU-1a, HU-1c, and HU-1d also inhibited HIV-1

at a 3 μM concentration. Number of compounds increased one

round HIV-1 replication, in accord with our previous study

showing activation of latent HIV-1 by PP1-targeting

Eﬀect of 1E7-03 Analogues on HIV-1 IIIB Replication.

We tested the eﬀect of the top three candidates in comparison

to 1E7-03 on the replication of T tropic HIV-1 IIIB in cultured

CEM T cells and also in primary peripheral blood

mononuclear cells (PBMCs). We focused on the analysis of

HIV-1 gag mRNA that occurs at the later stage of HIV-1

23

replication when unspliced HIV-1 mRNA is produced.

Compound HU-1a signiﬁcantly inhibited HIV-1 gag mRNA

production in CEM T cells (p < 0.01), similar to the eﬀect of

azidothymidine (AZT), used as positive control (Figure 5A).

In contrast, HU-1c had no eﬀect and HU-1d activated gag

production compared to the untreated control (Figure 5A).

Importantly, HU-1a showed stronger inhibition of HIV-1 gag

mRNA expression than 1E7-03 (p < 0.05) (Figure 5A). In

HIV-1 IIIB infected PBMCs, HU-1a also showed a trend

toward stronger reduction on HIV-1 gag mRNA expression

compared to 1E7-03, but no statistical signiﬁcance was reached

(Figure 5B). HU-1c showed no HIV-1 inhibition, whereas

HU-1d was less eﬃcient than HU-1a (Figure 5B). Next we

analyzed production of p24, a major core protein present in

22

compounds including SMAPP1. SMAPP1 increased PP1

activity in vitro and also induced expression of sds22, a PP1

shuttling subunit, potentially increasing nuclear PP1 pool and

22

inducing CDK9 phosphorylation on Ser-90. It is possible

that some of the HU compounds may have similar mechanism

of action. These compounds will be further analyzed elsewhere.

Biological screening of compounds HU-1a−HU-1k revealed

24

that compounds with phenyl substitution in R showed

HIV-1 virions, in supernatants collected from HIV-1 IIIB

infected CEM T cells that were subsequently treated with the

compounds. Levels of p24 were signiﬁcantly downregulated by

HU-1a compound similar to AZT compared to the untreated

sample (Figure 5C). Again, HU-1a was more eﬃcient than

1E7-03 (p < 0.01). In contrast, HU-1c and HU-1d had no

eﬀect when compared to the untreated control (Figure 5C).

Finally, we tested whether the inhibition of HIV-1 by HU-1a

and 1E7-03 required PP1. 293T cells were transfected with

plasmids expressing HIV-1 LTR luciferase reporter and HIV-1

Tat or with HIV-1 LTR luciferase reporter, HIV-1 Tat and

PP1. Transfected cells were treated with HU-1a or 1E7-03.

Coexpression of PP1 signiﬁcantly reduced HIV-1 transcription

inhibition by either compound (Figure 5D), conﬁrming that

2

superior HIV-inhibitory activity. Comparison of the activity

data for compounds HU-1e, HU-2h, and HU-6d suggested

that naphthyl substitution in R position resulted in reduced

2

HIV-1 inhibitory activity. In addition, the stronger HIV-1

inhibitory activities of HU-1a and HU-1b compounds

compared to HU-2e and HU-2f compounds suggested that

the urea linker was advantageous over the other linkers with an

amide bond. Most compounds HU-8a−HU-8e with p-

(

dimethylamino) styryl as R group showed HIV-1 activation

1.84−3.06-fold at 10 μM) instead of inhibition, implying that

1

the tertiary amine group in arylidene moiety may lead to the

loss of HIV-inhibitory activity. None of the compounds HU-

9

a−HU-9f showed any eﬀect, further conﬁrming the

J

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Figure 6. HU-1a primarily binds to the PP1 RVxF site as 1E7-03. (A) Eﬀect of HU-1a on the interaction of PP1:cdNIPP1 and their mutants

measured by Split NanoBit assay. HEK293T were transfected with the indicated plasmids for 24 h and treated with diﬀerent concentrations (1.3−

14 μM) of HU-1a for additional 6 h. Then NanoBiT luciferase activity was measured. Data are shown as percent of the untreated control. Shown

are the normalized mean ± SD of triplicates. (B) HU-1a binding to the RVxF-accommodating site of PP1 is shown. The residues close to the

ligand with the cutoﬀ distance of 8 Å were rendered by PyMOL software. HU-1a and its hydrogen bonding residues are shown as sticks. Hydrogen

bonds that HU-1a forms with residues Asp-242, Arg-261, and Leu-289 are shown as green dashes. (C) Eﬀect of 1E7-03, HU-1a, HU-1c, and HU-

1i on the interaction of PP1:cdNIPP1 measured by Split NanoBit assay. HEK293T were transfected with the plasmids of PP1 fused to the C-

terminus of the large bit and cdNIPP1 fused to the C-terminus of the small bit for 24 h and treated with diﬀerent concentrations (1.3−14 μM) of

the indicated compounds for 6 h. Then NanoBiT luciferase activity was measured. Data are shown as percent of the untreated control. Shown are

the normalized mean ± SD of triplicates. (D) Proposed molecular mechanism of HU-1a inhibition of HIV-1 transcription. HIV-1 Tat induces viral

transcription by interacting with PP1 RVxF-accommodating site, therefore promoting PP1 nuclear localization and recruitment of CDK9/cyclin T1

to HIV-1 TAR RNA to form the super elongation complex. Expression of cdNIPP1 or PP1-targeting compound treatment can disrupt the PP1:Tat

interaction and prevents recruitment of CDK9/cyclin T1 from 7SK RNA complex, blocking HIV-1 transcription and replication.

the inhibition was due to the targeting of PP1. Taken together,

HU-1a was the best all-around compound that eﬃciently

inhibited one round and multiple round HIV-1 replication and

suppressed the production of p24.

remained intact in human liver microsomes at 24 h, and 47% of

HU-1a versus 25% of 1E7-03 remained intact in mouse liver

microsomes at 24 h. We also found that HU-1a was more

stable in human liver microsomes than in mouse liver

microsomes (80% vs 47% of remaining at 24 h). Taken

together, HU-1a displayed improved metabolic stability in

mouse plasma and in both human and mouse liver microsomes

as compared with 1E7-03.

Metabolic Stability of HU-1a and 1E7-03. We next

compared metabolic stabilities of HU-1a and 1E7-03 that were

incubated in human or mouse plasma at 37 °C for 6 h.

Compounds were extracted as described in Methods and

analyzed by LC/FT-MS using a previously described

HU-1a Primarily Binds to the PP1 RVxF-Accommo-

dating Site. We used a competition assay (Split NanoBiT) to

determine whether HU-1a binds to the PP1 RVxF site in a

2

0

protocol. HU-1a was signiﬁcantly more stable in mouse

plasma than 1E7-03. The latter showed extensive decom-

position (>80%) in less than 30 min and 98% in 6 h (Figure

20

manner similar to 1E7-03 and 1E7-07. In this previously

developed assay, PP1 was fused to the C-terminus of the large

bit and cdNIPP1, a well-characterized PP1-binding central

domain of nuclear inhibitor of PP1 (cdNIPP1, residues 140−

225) was fused to the C-terminus of the small bit. We also

used binding deﬁcient mutants of cdNIPP1 including RVxF

motif mutant (cdNIPP1 RATA, V201A/F203A mutation) and

the helix motif mutant (cdNIPP1 mut, D164A/T171A/

N174A/K175A/I177A mutations). In addition, we used

5

E). In contrast, >95% HU-1a remained intact after 6 h

incubation (Figure 5E). In accord with our previous study,

both HU-1a and 1E7-03 were stable in human plasma during

6

h incubation (Figure 5E). We also tested metabolic stability

of HU-1a and 1E7-03 in human and mouse liver microsomes

Figure 5F). In both human and mouse liver microsomes, HU-

a was signiﬁcantly more stable than 1E7-03 during 24 h

incubation. About 80% of HU-1a versus 11% of 1E7-03

(

1

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modiﬁed PP1 mutated within the C-terminal groove (PP1

mut, Y70W/L73Y/G274E/A299E mutations). We previously

showed that cdNIPP1 RVxF mutation strongly reduced the

binding of cdNIPP1 to PP1 (70-fold reduction), whereas

mutations in cdNIPP1 helix motif and PP1 C-terminal groove

had moderate eﬀect (6-fold and 17-fold reduction, correspond-

CDK9/cyclin T1 to HIV-1 TAR RNA and triggers HIV-1

transcription elongation. CDK9 phosphorylates negative

elongation factor (NELF) and the DRB-sensitivity inducing

complex (DSIF/Spt4/Spt5), thus promoting the release of

NELF and also phosphorylates Ser-2 residues in the C-

terminal domain of RNAPII facilitating the formation of super

2

0

25

ingly). These previous results suggested that the RVxF motif

was the strongest contributor to the PP1:cdNIPP1 interaction,

elongation complex. HIV-1 Tat protein binds to the RVxF-

14

accommodating site of PP1 and shuttles PP1 into nucleus.

11

consistent with the NIPP1:PP1 holoenzyme structure. To

test the eﬀect of HU-1a on PP1:cdNIPP1 binding, HEK293T

cells were co-transfected with the indicated NanoBiT plasmid

pairs for 24 h and then treated with diﬀerent concentrations

PP1 dephosphorylates the Thr-186 of cyclin-dependent kinase

9 (CDK9), promoting recruitment of CDK9/cyclinT1 from

26

7SK small nuclear ribonuclear protein (snRNP) complex

(Figure 6D). PP1 can also dephosphorylate CDK9 Ser-175

26

(

1.3−14 μM) of HU-1a for an additional 6 h. HU-1a strongly

and Ser-90 residues. Expression of cdNIPP1 as part of HIV-1

genome or in a stable cell line locks CDK9/cyclin T1 within

7SK RNA complex and blocks HIV-1 transcription and

competed with PP1:cdNIPP1 binding (IC₅₀= 7.5 μM).

Mutation of cdNIPP1 with the RVxF motif resulted in

signiﬁcant reduction of the binding (not shown) which was

weakly aﬀected by HU-1a (IC₅₀= 34.4 μM) (Figure 6A),

suggesting that HU-1a primarily binds to the RVxF site of PP1.

HU-1a competed with PP1:cdNIPP1 helix mut binding (IC₅₀

27

replication. Because of their structural similarity, HU-1a is

likely to prevent the shuttling of PP1 into the nucleus, similar

to 1E7-03. The compound might also mimic cdNIPP1,

preventing HIV-1 Tat from PP1 binding and blocking

CDK9/cyclin T1 recruitment from 7SK snRNP (Figure 6D).

=

12.7 μM) and PP1 C-terminal groove mut:cdNIPP1 (IC =

50

9

.2 μM), and these IC s were close to the PP1:cdNIPP1

50

^c^o^m^p^e^tⁱ^tⁱ^oⁿ⁽^I^C⁵0 = 7.5) (Figure 6A). These observations

suggest that neither the helix binding pocket of PP1 nor the

PP1 C-terminal groove contributes strongly to the binding of

HU-1a. These results are in agreement with our previous

ﬁndings that 1E7-03 and 1E7-07 primarily interact with the

CONCLUSION

■

We report the development of a novel analogue HU-1a that

was structurally-inspired from a previously reported PP1-

targeting HIV-1 transcription inhibitor, 1E7-03. HU-1a

exhibited signiﬁcantly improved metabolic stability and

superior all-around HIV-1 inhibitory activity. Moreover, HU-

20

PP1 RVxF-accommodating site. These results also suggest

that both HU-1a and 1E7-03 weakly bind to the C-terminal

groove and NIPP1 helix pocket of PP1, further explaining the

protection by 1E7-03 of Arg-187 and Arg-188 residues from

being painted by ELNLLB. To corroborate HU-1a binding to

PP1, we docked HU-1a into the RVxF site of PP1 (Figure 6B).

HU-1a aligned well within the RVxF-accommodating site of

PP1 and formed several hydrogen bonds. One bond was

formed between the −NH group in the cyclopenta[b]-

quinolone core of HU-1a and Arg-261 (Figure 6B). This

bond was not formed when 1E7-03 was docked to PP1 (Figure

1

a binds to PP1 in a manner similar to 1E7-03 and may share

Asp-242 and Leu-289 residues within the RVxF-accommodat-

ing site of PP1. This new analogue is a promising HIV-1

transcription inhibitor and will be used as a lead molecule in

subsequent studies. Our study indicates that the RVxF-binding

site of PP1 may serve as a target for future design of novel

drugs to inhibit HIV-1 transcription.

EXPERIMENTAL PROCEDURES

■

2

C). Three additional hydrogen bonds were formed between

the urea motif of HU-1a with Asp-242 and Leu-289 (Figure

B). These bonds were also observed from 1E7-03 docking

General Methods. All the reagents and solvents were

purchased from commercial sources such as Sigma-Aldrich and

Oakwood Chemical, unless otherwise speciﬁed. Chemical

reactions were monitored using thin-layer chromatography

(TLC) on Whatmann silica gel 60 Å plates with a ﬂuorescent

indicator and visualized using a UV lamp (254 nm).

Puriﬁcation of compounds was carried out using column

chromatography with Merck silica-gel (60−120 mesh) as the

6

being formed between PP1 and the N-[(ethylamino)carbonyl]-

acetamide chain of 1E7-03 (Figure 2C). Thus, HU-1a and

1E7-03 interact with the RVxF-accommodating site of PP1 by

sharing common binding residues Asp-242 and Leu-289.

Finally, we compared the eﬀects of HU-1a and 1E7-03 to the

eﬀects of less inhibitory HU-1c and HU-1i compounds using

the Split NanoBit system (Figure 6C). Both HU-1c (IC = 3.9

1

13

stationary phase. H NMR and C NMR data were recorded

on a Bruker AVANCE III ( H 400 MHz, C 100 MHz)

spectrometer with CDCl₃or DMSO-d₆as solvents.

1

13

5

0

1

μM) and HU-1i (IC = 3.4 μM) showed stronger competition

H

50

with PP1:cdNIPP1 binding than HU-1a (IC₅₀= 6.5 μM) and

E7-03 (IC₅₀= 6.8 μM) (Figure 6C). This observation

chemical shifts were referenced to CDCl at 7.26 ppm and

3

1

3

1

for DMSO-d at 2.50 and 3.31 ppm. C chemical shifts were

6

suggests that the strength of the PP1 binding does not predict

well the extent of HIV-1 inhibition. Our previous study

showed that incorporation of strong PP1 binding RVTF motif

in place of QVCF motif within HIV-1 Tat blocked HIV-1

transcription, suggesting that intrinsically weak PP1 binding is

referenced to CDCl at 77 ppm and DMSO-d at 39.8 ppm

3

6

1

and obtained with H decoupling. Multiplicities are abbre-

viated as follows: singlet (s), doublet (d), triplet (t), quartet

(q), doublet−doublet (dd), doublet−doublet−doublet (ddd),

multiplet (m), and broad singlet (bs). Purities were

determined by a LC-20AD nano HPLC system (Shimadzu

Corporation, Columbia, MD) coupled to LTQ XL Orbitrap

mass spectrometer (Thermo Fisher Scientiﬁc), and all

compounds were conﬁrmed to have ≥95% purity (Figure S2).

General Procedures for the Synthesis of Compounds HU-

1a to HU-1k. Triethyl amine (0.66 mmol) and isocynate (0.34

mmol) were added into intermediate 5 (0.20 mmol) in

anhydrous DMF (5 mL). The reaction contents were allowed

14

required for HIV-1 transcription. We also found that 1E7-07

20

compound that bound stronger to PP1 was not able to

inhibit HIV-1 but, instead, activated HIV-1 replication (data

not shown). Thus, optimal PP1 binding and, perhaps, optimal

cellular localization is required for the development of optimal

HIV-1 inhibitors.

Our ﬁndings are summarized in the model shown in Figure

D. HIV-1 transcription is induced by Tat protein that recruits

6

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to stir at RT overnight (approx. 14−16 h). The crude reaction

mixture was diluted with dichloromethane (15 mL), and water

boxamide (HU-1c). HU-1c was obtained as yellow solid

1

(0.038 g, 29% yield). H NMR (400 MHz, DMSO-d ) δ :

6

H

(

20 mL) was added. The organic layer was separated, washed

8.75−8.73 (m, 1H), 8.49 (s, 1H), 8.01−7.99 (d, J = 8.0 Hz,

1H), 7.87−7.85 (d, J = 8.0 Hz, 1H), 7.70−7.67 (m, 2H),

7.48−7.46 (m, 1H), 7.30−7.21 (m, 4H), 7.06−7.01 (m, 3H),

6.17−6.16 (m, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.48−3.35 (m,

with brine solution (15 mL), dried over anhydrous sodium

sulfate, ﬁltered, and concentrated using a rotary evaporator.

The crude product was then puriﬁed using silica gel (60−100

mesh) ﬂash column chromatography. Gradient elution using

1

3

4H), 3.18−3.17 (m, 4H), 2.21 (s, 3H). C NMR (100 MHz,

DMSO-d ) δ : 166.6, 162.4, 155.9, 149.2, 149.2, 148.3, 139.1,

1

%−10% methanol in dichloromethane furnished the desired

product.

The yield of HU-1 series compounds was low (see below).

6

C

138.5, 138.4, 133.5, 130.2, 130.2, 129.5, 129.5, 129.3, 126.6,

125.6, 124.9, 124.4, 122.9, 118.3, 113.4, 112.4, 56.0, 55.9, 28.5,

+

Analysis of the crude reaction mixture by thin-layer

chromatography showed complete consumption of the starting

amine along with the presence of co-eluting undesired product.

Changing the stoichiometric ratios of the reactants did not

improve the ratio of undesired:desired product. Changing the

mobile phase during elution did not oﬀer any advantage during

puriﬁcation. Multiple attempts of silica gel ﬂash column

chromatography were required to get a pure sample. Poor

solubility of the product during chromatographic puriﬁcation

also posed a challenge. Thus, the isolated yield of the product

remained low. We also attempted to subject the crude product

to subsequent reactions. However, it gave poor yields and a

mixture of products in the subsequent steps. We were unable

to determine the chemical identity of the impurity because of

the inability to isolate them without desired amount. We

examined if the use of other solvents, such as CH Cl , would

26.8, 20.7. HRMS (ESI) calculated for C H N O [M + H]

3

2

32

4

m/z, 537.2502; found, 537.2515.

(E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(3-(4-

methoxyphenyl)ureido)ethyl)-2,3-dihydro-1H-cyclopenta-

[b]quinoline-9-carboxamide (HU-1d). HU-1d was obtained

1

as red solid (0.035 g, 29% yield). H NMR (400 MHz, DMSO-

d ) δ : 8.76−8.73 (t, 1H), 8.41 (s, 1H), 8.01−7.99 (d, J = 8.1

6

H

Hz, 1H), 7.88−7.85 (m, 1H), 8.03 7.71−7.67 (m, 2H), 7.49−

7.45 (m, 1H), 7.33−7.30 (m, 2H), 7.29−7.21 (m, 2H), 7.06−

7.04 (d, J = 8.0 Hz, 1H), 6.82−6.80 (m, 1H), 6.13−6.11 (m,

1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.68 (s, 3H), 3.48−3.47 (m,

1

3

4H), 3.21−3.17 (m, 4H). C NMR (100 MHz, DMSO-d )

C

6

δ : 166.6, 162.4, 156.1, 154.4, 149.2, 149.2, 148.3, 139.1,

138.5, 134.1, 133.5, 130.2, 129.5, 129.3, 126.6, 125.6, 124.9,

124.4, 122.9, 119.9, 114.3, 113.5, 112.4, 56.0, 55.9, 55.6, 28.5,

+

26.8. HRMS (ESI) calculated for C H N O [M + H] m/z,

2

32 32

4

5

suppress the impurity formation. However, the reaction was

sluggish in CH Cl and failed to yield appreciable conversion

after 24 h. Other solvents like DMSO led to problems during

isolation and puriﬁcation of the desired product. Thus, DMF

was considered to be the optimal solvent for this reaction.

553.2451; found, 553.2466.

(E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(3-(naphthalen-2-

yl)ureido)ethyl)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-

carboxamide (HU-1e). HU-1e was obtained as yellow solid

2

1

(0.028 g, 17% yield). H NMR (400 MHz, DMSO-d ) δ :

6

H

(

E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(3-(4-

8.90 (s, 1H), 8.88−8.85 (t, 1H), 8.04 (s, 1H), 7.82−7.80 (d, J

= 8.0 Hz, 1H), 7.78−7.74 (m, 1H), 7.72−7.69 (m, 1H), 7.67−

7.66 (m, 5H), 7.49−7.43 (m, 3H), 7.42−7.40 (t, 1H), 7.22 (s,

1H), 7.16−7.14 (d, J = 8.0 Hz, 1H), 6.34−6.32 (m, 1H), 3.81

(s, 6H), 3.58−3.49 (m, 4H), 3.22−3.21 (m, 2H), 3.12−3.11

(

triﬂuoromethyl)phenyl)ureido)ethyl)-2,3-dihydro-1H-

cyclopenta[b]quinoline-9-carboxamide (HU-1a). HU-1a was

obtained as light yellow solid (0.035 g, 21% yield). H NMR

400 MHz, DMSO-d ) δ : 9.09 (s, 1H), 8.79−8.76 (t, 1H),

.01−7.99 (d, J = 8.0 Hz, 1H), 7.96−7.98 (d, J = 8.0 Hz, 1H),

.88−7.70 (m, 2H), 7.68−7.62 (m, 2H), 7.58−7.50 (m, 2H),

.47−7.41 (m, 1H), 7.25−7.00 (m, 2H), 7.03−7.00 (d, 1H),

.40−6.37 (t, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.51−3.50 (m,

H), 3.44−3.41 (m, 2H), 3.19−3.17 (m, 4H). C NMR (100

MHz, DMSO-d ) δ : 166.7, 162.4, 155.5, 149.2, 148.3, 144.7,

1

(

6

H

1

3

8

7

6

2

(m, 2H). C NMR (100 MHz, DMSO-d ) δ : 166.7, 162.4,

6 C

155.9, 149.2, 149.1, 148.3, 139.1, 138.7, 138.5, 134.3, 133.6,

130.1, 129.5, 129.2, 128.7, 127.9, 127.2, 126.7, 126.6, 125.6,

124.9, 124.4, 124.0, 122.7, 119.9, 113.5, 113.1, 112.4, 56.0,

55.9, 28.5, 26.8. HRMS (ESI) calculated for C H N O [M +

13

3

5

32

4

+

H] m/z, 573.2502; found, 573.2513.

6

C

1

39.1, 138.5, 133.5, 130.2, 129.5, 129.2, 126.6, 126.4, 126.4,

25.6, 124.9, 124.4, 123.8, 122.8, 121.6, 121.3, 117.7, 113.5,

(E)-N-(2-(3-Cyclopentylureido)ethyl)-3-(3,4-dimethoxy-

benzylidene)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-car-

boxamide (HU-1f). HU-1f was obtained as yellow solid (0.025

12.4, 56.0, 55.9, 28.5, 26.8. HRMS (ESI) calculated for

+

1

C H F N O [M + H] m/z, 591.2219; found, 591.2233.

g, 18% yield). H NMR (400 MHz, DMSO-d ) δ : 8.70 (s,

3

2

29

3

4

6

H

(

E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(3-(4-

1H), 8.02−8.00 (d, J = 8.0 Hz, 1H), 7.86−7.84 (d, J = 8.0 Hz,

1H), 7.72−7.69 (m, 2H), 7.54−7.50 (m, 1H), 7.26−7.23 (m,

2H), 7.07−7.05 (d, J = 8.0 Hz, 1H), 6.01−5.99 (d, J = 8 Hz,

1H), 5.80 (s, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.41−3.39 (m,

2H), 3.26−3.21 (m, 7H), 1.77−1.75 (m, 2H), 1.60−1.48 (m,

ﬂuorophenyl)ureido)ethyl)-2,3-dihydro-1H-cyclopenta[b]-

quinoline-9-carboxamide (HU-1b). HU-1b was obtained as

light yellow solid (0.04 g, 25% yield). H NMR (400 MHz,

1

DMSO-d ) δ : 8.76−8.73 (t, 1H), 8.66 (s, 1H), 8.00−7.98 (d,

6

H

1

3

J = 8.0 Hz, 1H), 7.86−7.84 (d, J = 8.0 Hz, 1H), 7.69−7.67 (m,

4H), 1.32−1.29 (m, 2H). C NMR (100 MHz, DMSO-d )

6

2

3

(

H), 7.47−7.39 (m, 3H), 7.23−7.20 (m, 2H), 7.07−7.03 (m,

H), 6.22−6.19 (t, 1H), 3.83 (s, 3H), 3.80 (s, 3 H), 3.48−3.47

δ : 166.5, 162.4, 162.1, 158.3, 149.2, 149.2, 148.3, 139.1,

C

138.5, 133.5, 130.2, 129.6, 129.3, 126.6, 125.6, 124.9, 124.4,

122.9, 113.4, 112.4, 56.0, 55.9, 33.4, 28.6, 26.8, 23.6. HRMS

1

3

m, 2H), 3.36−3.35 (m, 2H), 3.17−3.16 (m, 4H).). C NMR

+

100 MHz, DMSO-d ) δ : 166.7, 1162.4, 162.1, 158.5, 156.2,

(ESI) calculated for C H N O [M + H] m/z, 515.2665;

6

C

30 34

4

1

55.9, 149.2, 149.2, 148.3, 139.1, 138.5, 137.4, 133.5, 130.2,

29.5, 129.3, 126.6, 125.6, 124.9, 124.4, 122.9, 119.8, 119.7,

found, 515.2665.

(E)-N-(2-(3-Cyclobutylureido)ethyl)-3-(3,4-dimethoxyben-

zylidene)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-carbox-

amide (HU-1g). HU-1g was obtained as yellow solid (0.03 g,

15.6, 115.4, 113.4, 112.4, 56.0, 55.9, 28.5, 26.8. HRMS (ESI)

+

calculated for C H FN O [M + H] m/z 541.2251; found,

3

1

29

4

1

5

41.2262.

E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(3-(p-tolyl)-

ureido)ethyl)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-car-

25% yield). H NMR (400 MHz, DMSO-d ) δ : 8.70−8.68 (t,

6

H

(

1H), 8.0−7.99 (d, J = 8.0 Hz, 1H), 7.85−7.83 (d, J = 8.0 Hz,

1H), 7.72−7.68 (m, 2H), 7.53−7.50 (m, 1H), 7.26−7.23 (m,

M

https://dx.doi.org/10.1021/acsinfecdis.0c00511

ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

pubs.acs.org/journal/aidcbc

Article

1

3

2

1

3

1

5

H), 7.06−7.04 (d, J = 8.0 Hz, 1H), 6.29−6.27 (d, J = 8.0 Hz,

H), 5.86−5.85 (m, 1H), 4.07−4.05 (m, 1H), 3.84 (s, 3H),

.81 (s, 3H), 3.44−3.41 (m, 2H), 3.24−3.21 (m, 4H), 3.17−

.15 (m, 2H), 2.14−2.10 (m, 2H), 1.80−1.75 (m, 2H), 1.57−

3H). C NMR (100 MHz, DMSO-d ) δ : 182.8, 166.5, 162.3,

6 C

162.1, 158.6, 149.2, 149.2, 148.3, 141.4, 139.1, 138.5, 133.5,

130.2, 129.5, 129.3, 127.7, 126.6, 125.6, 124.9, 124.4, 122.9,

113.5, 112.4, 56.0, 55.9, 34.6, 28.6, 26.8, 17.2, 16.1. HRMS

(ESI) calculated for C H N O [M + H] m/z, 475.2345;

found, 475.2351.

General Procedures for the Synthesis of Compounds HU-

2a−HU-2j. The triethyl amine (0.62 mmol) and T P (0.62

mmol) were added to a stirred mixture of acids and immediate

5 (0.24 mmol) in dichloromethane (5 mL) at 0° C. The

reaction mixture was allowed to stir at RT overnight (approx.

14−16 h). The crude reaction mixture was diluted with

dichloromethane (15 mL), and water (20 mL) was added. The

organic layer was separated, washed with brine solution (20

mL), dried over anhydrous sodium sulfate, ﬁltered, and

concentrated using a rotary evaporator. The crude product

was then puriﬁed using silica gel (60−120 mesh) ﬂash column

chromatography. Gradient elution using 30%−100% EtOAc in

hexanes furnished the desired product.

1

3

+

.53 (m, 2H). C NMR (100 MHz, DMSO-d ) δ : 166.5,

6

C

27 30

4

62.4, 157.5, 149.1, 148.3, 139.1, 138.5, 133.5, 130.2, 129.6,

29.3, 126.6, 125.6, 124.9, 124.4, 122.9, 113.4, 112.4, 58.6,

6.0, 55.9, 45.2, 45.0, 31.7, 28.6, 26.8, 17.16, 14.7. HRMS

3

+

(

ESI) calculated for C H N O [M + H] m/z, 501.2502;

29

32

4

found, 501.2512.

(

E)-N-(2-(3-Cyclopropylureido)ethyl)-3-(3,4-dimethoxy-

benzylidene)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-car-

boxamide (HU-1h). HU-1h was obtained as yellow solid (0.04

g, 34% yield). H NMR (400 MHz, DMSO-d ) δ : 8.72 (s,

1

6

H

1

H), 8.02−8.00 (d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.86−7.84

(

d, J = 8.0 Hz, 1H), 7.72−7.69 (m, 2H), 7.54−7.51 (t, 1H),

7

1

3

0

.26−7.23 (m, 2H), 7.06−7.04 (d, J = 8.0 Hz, 1H), 6.29 (s,

H), 6.04−6.01 (m, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.29−

.21 (m, 4H), 2.88−2.73 (m, 4H), 2.40−2.39 (m, 1H), 0.57−

13

.55 (m, 2H), 0.35 (m, 2H). C NMR (100 MHz, DMSO-d )

(E)-N-(2-(Cyclopropanecarboxamido)ethyl)-3-(3,4-dime-

thoxybenzylidene)-2,3-dihydro-1H-cyclopenta[b]quinoline-

9-carboxamide (HU-2a). HU-2a was obtained as yellow solid

6

δ_C: 166.6, 162.8, 162.4, 159.9, 159.4, 149.2, 149.2, 148.3,

1

2

39.1, 138.5, 133.5, 130.2, 129.6, 129.3, 126.6, 125.5, 124.9,

24.4, 122.9, 113.5, 112.4, 56.0, 55.9, 36.2, 31.2, 28.6, 26.8,

2.9, 22.8, 7.1, 6.9. HRMS (ESI) calculated for C H N O

4

1

(0.035 g, 31% yield). H NMR (400 MHz, DMSO-d ) δ :

6

H

8.73−8.70 (t, 1H), 8.17−8.14 (t, 1H), 8.01−7.99 (d, J = 8 Hz,

1H), 7.85−7.83 (d, J = 8 Hz, 1H), 7.72−7.68 (m, 2H), 7.54−

7.50 (m, 1H), 7.26−7.23 (m, 2H), 7.06−7.04 (d, J = 8 Hz,

1H) 3.84 (s, 3H), 3.80 (s, 3H), 3.55−3.34 (m, 4H), 3.25−3.20

(m, 6H), 1.59 (m, 1H), 0.69−0.65 (m, 4H). C NMR (100

MHz, DMSO-d ) δ : 173.3, 166.5, 162.4, 149.2, 149.2, 148.3,

2

8

30

4

+

[

M + H] m/z, 487.2345; found, 487.2343.

E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(3-(furan-2-

(

ylmethyl)ureido)ethyl)-2,3-dihydro-1H-cyclopenta[b]-

quinoline-9-carboxamide (HU-1i). HU-1i was obtained as

yellow solid (0.036 g, 22% yield). H NMR (400 MHz,

1

3

1

6

C

DMSO-d ) δ : 8.72−8.69 (t, 1H), 8.02−8.00 (d, J = 8.0 Hz,

139.0, 138.5, 133.6, 130.2, 129.6, 129.3, 126.6, 125.6, 124.9,

124.4, 122.9, 113.5, 112.4, 56.0, 55.9, 38.9, 28.6, 26.9, 14.2, 6.7.

6

C

1

7

H), 7.86−7.84 (d, J = 8.0 Hz, 1H), 7.72−7.68 (m, 2H),

.54−7.49 (m, 2H), 7.26−7.23 (m, 2H), 7.07−7.05 (d, J = 8.0

+

HRMS (ESI) calculated for C H N O [M + H] m/z,

2

8

29

3

4

Hz, 1H), 6.46−6.43 (m, 2H), 6.19−6.04 (m, 2H), 4.21−4.20

472.2236; found, 472.2247.

(

2

d, J = 4 Hz, 2H), 3.84 (s, 3H), 3.81 (s, 3H), 3.42−3.40 (m,

(E)-3-(4-(Dimethylamino)benzylidene)-N-(2-(furan-3-

carboxamido)ethyl)-2,3-dihydro-1H-cyclopenta[b]-

quinoline-9-carboxamide (HU-2b). HU-2b was obtained as

13

H), 3.27−3.20 (m, 6H). C NMR (100 MHz, DMSO-d )

6

δ : 166.5, 162.4, 158.3, 154.1, 149.2, 149.2, 142.3, 139.1,

C

1

38.5, 133.5, 130.2, 129.6, 126.6, 125.6, 124.9, 124.4, 122.9,

brown solid (0.025 g, 41% yield). H NMR (400 MHz,

13.5, 112.4, 110.8, 106.6, 56.0, 55.9, 36.9, 28.6, 26.8. HRMS

DMSO-d ) δ : 7.97 (s, 1H), 7.90−7.88 (m, 2H), 7.66−7.64

6

H

+

(

ESI) calculated for C H N O [M + H] m/z, 527.2294;

(m, 4H), 7.57−7.47 (m, 2H), 7.26−7.21 (m, 2H), 6.74−6.67

30

4

5

13

found, 527.2296.

(m, 1H), 3.79 −3.74 (m, 2H), 3.21−2.71 (m, 12H). C NMR

(100 MHz, DMSO-d ) δ : 163.4, 162.1, 150.1, 149.9, 145.2,

(

E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(3-(2,2,2-

6

C

triﬂuoroethyl)ureido)ethyl)-2,3-dihydro-1H-cyclopenta[b]-

quinoline-9-carboxamide (HU-1j). HU-1j was obtained as

yellow solid (0.035 g, 21% yield). H NMR (400 MHz, DMS0-

143.7, 133.9, 131.4, 131.2, 129.5, 129.1, 127.8, 127.4, 126.7,

126.5, 126.1, 124.6, 123.9, 123.5, 122.5, 112.0, 108.6, 45.3,

40.8, 40.2, 40.2, 39.8. HRMS (ESI) calculated for C H N O

1

2

9

28

4

3

+

d ) δ : 8.73−8.71 (t, 1H), 8.02−8.00 (d, J = 8.0 Hz, 1H),

[M + H] m/z, 481.2240; found, 481.2031.

6

H

7

6

3

.86−7.83 (m, 1H), 7.73−7.69 (m, 2H), 7.54−7.50 (m, 1H),

.27−7.24 (m, 2H), 7.07−7.11 (m, 1H), 6.70−6.67 (t, 1H),

.28−6.25 (t, 1H), 3.85 (s, 3H), 3.83−3.81 (m, 5H), 3.41−

(E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(thiophene-3-

carboxamido)ethyl)-2,3-dihydro-1H-cyclopenta[b]-

quinoline-9-carboxamide (HU-2c). HU-2c was obtained as

13

1

.42 (m, 2H), 3.31−3.21 (m, 6H). C NMR (100 MHz,

yellow solid (0.045 g, 70% yield). H NMR (400 MHz,

DMSO-d ) δ : 166.6, 162.4, 162.1, 157.9, 149.2, 149.2, 148.3,

DMSO-d ) δ : 8.13−8.11 (d, J = 8.00 Hz, 1H), 7.69−7.65 (m,

6

C

6

H

1

5

41.1, 139.1, 138.5, 133.5, 130.2, 129.8, 129.6, 129.3, 127.9,

27.1, 126.6, 125.5, 124.9, 124.4, 124.3, 122.9, 113.4, 112.4,

1H), 7.54−7.50 (m, 2H), 7.49−7.47 (m, 2 H), 7.35−7.34 (m,

1H), 7.23−7.21 (m, 1H), 7.18−7.15 (m, 2H), 6.94−6.92 (d, J

= 8 Hz, 1H), 3.96 (s, 3H), 3.93 (s, 3H), 3.60−3.52 (m, 4H),

6.0, 55.9, 41.2, 40.8, 31.4, 28.6, 26.8, 26.4, 22.5. HRMS (ESI)

+

13

calculated for C H F N O [M + H] m/z, 529.2063; found,

3.31−3.20 (m, 2 H), 3.11−3.09 (m, 2H). C NMR (100

2

7

27

3

4

5

29.2062.

E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(3-ethylureido)-

MHz, DMSO-d ) δ : 166.8, 166.0, 162.5, 149.0, 148.9, 137.4,

6

C

(

135.7, 135.5, 133.0, 129.8, 129.5, 127.2, 126.9, 126.8, 126.8,

125.9, 123.7, 122.7, 113.3, 112.9, 112.2, 110.4, 108.9, 55.9,

55.9, 46.5, 46.2, 26.9, 26.7. HRMS (ESI) calculated for

ethyl)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-carboxa-

mide (HU-1k). HU-1k was obtained as gray solid (0.05 g, 30%

1

+

yield). H NMR (400 MHz, DMSO-d ) δ : 8.71 (s, 1H),

C H N O S [M + H] m/z, 514.1801; found, 514.1723.

6

H

29 27

3

4

8

.01−7.99 (d, 1H), 7.86−7.84 (d, J = 8.0 Hz, 1H), 7.71−7.68

(E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(thiophene-2-

carboxamido)ethyl)-2,3-dihydro-1H-cyclopenta[b]-

quinoline-9-carboxamide (HU-2d). HU-2d was obtained as

(

m, 2H), 7.53−7.50 (t, 1H), 7.26−7.23 (m, 2H), 7.06−7.04

d, J = 8.0 Hz, 1H), 5.99−5.97 (m, 2H), 3.84 (s, 3H), 3.80 (s,

1

3

H), 3.40−3.38 (m, 6H), 3.16−2.86 (m, 4H), 1.00−0.97 (t,

yellow solid (0.035 g, 31% yield). H NMR (400 MHz,

N

https://dx.doi.org/10.1021/acsinfecdis.0c00511

ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

pubs.acs.org/journal/aidcbc

Article

DMSO-d ) δ : 8.80 (s, 1H), 8.61 (s, 1H), 8.01−7.99 (d, J = 8

133.5, 132.6, 130.8, 130.2, 129.2, 129.0, 128.5, 127.9, 127.8,

127.7, 126.8, 126.4, 125.5, 124.4, 123.9, 123.4, 122.7, 112.7,

111.2, 55.9, 55.9, 41.1, 40.2, 28.5, 26.5. HRMS (ESI)

6

H

Hz, 1H), 7.85−7.83 (d, J = 8 Hz, 1H), 7.72−7.68 (m, 4H),

7

.46−7.42 (t, 1H), 7.26 (m, 2H), 7.23−7.17 (m, 1H), 7.06−

+

7

.04 (d, J=8 Hz, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.57−3.53

calculated for C H N O [M + H] m/z, 558.2393; found,

3

5

31

3

4

(

m, 4H), 3.19 (m, 4H). ¹³C NMR (100 MHz, DMSO-d ) δ :

558.2385.

6

C

1

2

66.6, 162.4, 161.8, 149.2, 149.2, 148.3, 140.5, 139.0, 138.5,

33.6, 131.2, 130.2, 129.5, 129.3, 128.5, 128.3, 126.6, 125.6,

24.9, 124.4, 122.9, 113.5, 112.4, 56.0, 55.9, 31.4, 28.6, 26.9,

(E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(nicotinamido)-

ethyl)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-carboxa-

mide (HU-2i). HU-2i was obtained as yellow solid (0.03 g,

+

1

2.5. HRMS (ESI) calculated for C H N O S [M + H] m/

65% yield). H NMR (400 MHz, CDCl ) δ : 9.08 (s, 1H),

29

27

3

4

3

H

z, 514.1801; found, 514.1809.

8.66 (d, J = 4.4 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 8.00 (d, J =

8.5 Hz, 1H), 7.78−7.66 (m, 2H), 7.66−7.53 (m, 2H), 7.32

(dd, J = 7.5, 4.2 Hz, 3H), 7.12 (d, J = 8.4 Hz, 1H), 7.05 (s,

1H), 6.87 (t, J = 9.8 Hz, 1H), 3.90 (s, 3H), 3.90 (s, 3H), 3.81

(

E ) - 3 - ( 3 , 4 - D i m e t h o x y b e n z y l i d e n e ) - N - ( 2 - ( 4 -

ﬂuorobenzamido)ethyl)-2,3-dihydro-1H-cyclopenta[b]-

quinoline-9-carboxamide (HU-2e). HU-2e was obtained as

yellow solid (0.035 g, 51% yield). H NMR (400 MHz,

1

13

(d, J = 5.2 Hz, 2H), 3.78 (d, J = 4.9 Hz, 2H), 2.95 (s, 4H). C

DMSO-d ) δ : 8.81 (s, 1H), 8.61 (s, 1H), 8.06 −7.91 (m,

NMR (100 MHz, CDCl ) δ : 26.6, 28.3, 31.6, 39.6, 41.1, 55.9,

6

H

3

C

3

H), 7.84 (d, J = 7.5 Hz, 1H), 7.69 (s, 2H), 7.45 (s, 1H), 7.29

111.2, 112.7, 122.8, 123.5, 124.3, 125.6, 126.3, 128.9, 129.3,

129.5, 130.0, 133.4, 134.9, 137.2, 137.3, 148.0, 148.8, 148.9,

152.2, 162.2, 166.1, 168.5. HRMS (ESI) calculated for

(

3

d, J = 25.4 Hz, 4H), 7.06 (d, J = 7.6 Hz, 1H), 3.85 (s, 3H),

.82 (s, 3H), 3.57 (s, 4H), 3.19 (s, 4H). C NMR (100 MHz,

13

+

DMSO-d ) δ : 166.6, 165.8, 165.6, 163.1, 162.4, 149.2, 149.2,

C H N O [M + H] m/z, 509.2189; found, 509.2180.

6

C

30 28

4

1

48.3, 139.0, 138.5, 133.6, 131.5, 131.4, 130.4, 130.3, 130.2,

29.6, 129.3, 126.5, 125.6, 124.9, 124.4, 122.9, 115.8, 115.5,

(E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(pyridazine-4-

carboxamido)ethyl)-2,3-dihydro-1H-cyclopenta[b]-

quinoline-9-carboxamide (HU-2j). HU-2j was obtained as

13.5, 112.4, 56.0, 55.9, 31.4, 28.5, 26.8, 22.5. HRMS (ESI)

+

1

calculated for C H FN O [M + H] m/z, 526.2142; found,

yellow solid (0.025 g, 60% yield). H NMR (400 MHz,

3

1

28

3

4

5

26.2149.

E ) - 3 - ( 3 , 4 - D i m e t h o x y b e n z y l i d e n e ) - N - ( 2 - ( 4 -

triﬂuoromethyl)benzamido)ethyl)-2,3-dihydro-1H-

CDCl ) δ : 9.62 (s, 1H), 9.29 (d, J = 5.2 Hz, 1H), 8.28 (s,

3

H

(

1H), 8.05 (d, J = 8.5 Hz, 1H), 7.90−7.83 (m, 1H), 7.75−7.68

(m, 2H), 7.64 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.4 Hz, 1H),

7.14 (dd, J = 21.0, 11.5 Hz, 3H), 6.91 (d, J = 8.4 Hz, 1H), 3.93

(s, 3H), 3.92 (s, 3H), 3.87 (s, 2H), 3.83 (s, 2H), 3.06 (s, 4H),

(

cyclopenta[b]quinoline-9-carboxamide (HU-2f). HU-2f was

1

obtained as yellow solid (0.035 g, 31% yield). H NMR (400

MHz, DMSO-d ) δ : 8.83 (s, 2H), 8.09 (d, J = 7.5 Hz, 2H),

1

3

2.82 (s, 1H). C NMR (100 MHz, CDCl ) δ : 26.7, 28.4,

6

H

3 C

8

(

.01 (d, J = 8.1 Hz, 1H), 7.86 (dd, J = 16.3, 8.0 Hz, 3H), 7.69

s, 2H), 7.45 (t, J = 7.1 Hz, 1H), 7.24 (d, J = 12.4 Hz, 2H),

.05 (d, J = 7.9 Hz, 1H), 3.83 (d, J = 13.7 Hz, 6H), 3.60 (s,

39.6, 41.8, 55.9, 55.9, 111.2, 112.7, 122.8, 123.8, 124.1, 125.8,

126.5, 129.1, 129.4, 129.9, 131.1, 133.5, 137.0, 137.2, 148.5,

148.8, 149.0, 151.8, 162.1, 163.7, 169.0. HRMS (ESI)

7

4

1

2

13

+

H), 3.19 (s, 4H). C NMR (100 MHz, DMSO-d ) δ : 166.6,

calculated for C H N O [M + H] m/z, 510.2141; found,

6

C

29 27

5

4

65.7, 162.4, 149.2, 149.2, 148.3, 138.9, 138.8, 138.5, 133.6,

31.7, 131.4, 130.2, 129.6, 129.3, 128.6, 126.5, 125.8, 125.8,

25.5, 124.9, 124.4, 123.1, 122.9, 113.5, 112.4, 56.0, 55.9, 39.4,

8.5, 26.9. HRMS (ESI) calculated for C H F N O [M +

510.2132.

General Procedures for the Synthesis of Compounds HU-

3a−HU-3c. Triethyl amine (0.62 mmol) and sulfonyl chloride

(0.62 mmol) were added to immediate 5 (0.20 mmol) in

dichloromethane (5 mL) at 0° C. The reaction mixture was

allowed to stir at RT for 3 h. The crude reaction mixture was

diluted with dichloromethane (15 mL), and water (20 mL)

was added. The organic layer was separated, washed with brine

solution (20 mL), dried over anhydrous sodium sulfate,

ﬁltered, and concentrated using a rotary evaporator. The crude

product was then puriﬁed by silica gel (60−120 mesh) ﬂash

column chromatography. Gradient elution using 60%−100%

EtOAc in hexanes furnished the desired product.

32

28

3

4

+

H] m/z, 576.2210; found, 576.2202.

E ) - 3 - ( 3 , 4 - D i m e t h o x y b e n z y l i d e n e ) - N - ( 2 - ( 4 -

triﬂuoromethoxy)benzamido)ethyl)-2,3-dihydro-1H-

(

cyclopenta[b]quinoline-9-carboxamidee (HU-2g). HU-2g

1

was obtained as yellow solid (0.040 g, 41% yield). H NMR

(

400 MHz, DMSO-d ) δ : 8.81 (s, 1H), 8.70 (s, 1H), 8.01 (d,

6 H

J = 8.3 Hz, 3H), 7.83 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 8.2 Hz,

H), 7.55−7.40 (m, 3H), 7.24 (d, J = 11.9 Hz, 2H), 7.05 (d, J

8.1 Hz, 1H), 3.83 (d, J = 13.7 Hz, 6H), 3.64−3.53 (m, 4H),

2

=

3

1

3

.19 (s, 4H). C NMR (100 MHz, DMSO-d ) δ : 166.6,

(E)-3-(3,4-Dimethoxybenzylidene)-N-(2-((4-ﬂuorophenyl)-

sulfonamido)ethyl)-2,3-dihydro-1H-cyclopenta[b]quinoline-

9-carboxamide (HU-3a). HU-3a was obtained as red solid

6

C

1

65.7, 162.4, 150.7, 149.2, 149.2, 148.3, 139.0, 138.5, 134.1,

33.6, 130.2, 130.0, 129.5, 129.3, 126.5, 125.5, 124.9, 124.4,

22.9, 121.4, 121.1, 119.2, 113.5, 112.4, 56.0, 55.9, 39.4, 28.5,

1

(0.065 g, 77% yield). H NMR (400 MHz, CDCl ) δ : 9.23 (s,

3

H

+

2

6.8. HRMS (ESI) calculated for C H F N O [M + H] m/

1H), 8.83 (d, J = 3.5 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H), 8.00

(s, 1H), 7.80 (s, 1H), 7.57−7.47 (m, 2H), 7.34 (s, 2H), 7.17

(s, 2H), 6.94 (s, 3H), 4.02−3.91 (m, 5H), 3.80 (s, 8H), 3.46

32

28

3

5

z, 592.2059; found, 592.2052.

(E)-N-(2-(2-Naphthamido)ethyl)-3-(3,4-dimethoxybenzyli-

1

3

dene)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-carboxa-

(s, 6H), 2.85 (s, 2H). C NMR (100 MHz, CDCl ) δ : 165.9,

3 C

mide (HU-2h). HU-2h was obtained as yellow solid (0.02 g,

165.7, 163.4, 161.0, 150.1, 149.2, 137.2, 131.2, 130.1, 129.9,

129.5, 127.7, 125.9, 124.4, 124.1, 117.0, 116.8, 113.4, 112.4,

56.1, 55.9, 42.5, 28.6, 27.3, 27.1. HRMS (ESI) calculated for

1

5

8

5% yield). H NMR (400 MHz, DMSO-d ) δ : 8.36 (s, 1H),

.01 (d, J = 8.4 Hz, 1H), 7.89−7.81 (m, 4H), 7.76 (d, J = 8.2

6

H

+

Hz, 1H), 7.65 (s, 1H), 7.59−7.52 (m, 4H), 7.48 (t, J = 6.7 Hz,

H), 7.33 (s, 1H), 7.10 (d, J = 6.9 Hz, 1H), 7.04 (s, 1H), 6.88

d, J = 8.4 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H), 3.02 (dd, J =

C H FN O S [M + H] m/z, 562.1812; found, 562.1812.

3

0

28

3

5

1

(

(E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(pyridine-3-

sulfonamido)ethyl)-2,3-dihydro-1H-cyclopenta[b]quinoline-

9

2

.1, 4.5 Hz, 2H), 2.96 (d, J = 7.4 Hz, 1H), 2.91 (d, J = 3.7 Hz,

H), 1.29 (dd, J = 16.6, 9.3 Hz, 2H). C NMR (100 MHz,

9-carboxamide (HU-3b). HU-3b was obtained as red solid

13

1

(0.06 g, 75% yield). H NMR (400 MHz, DMSO-d ) δ : 9.23

6

H

DMSO-d ) δ : 168.5, 168.3, 148.9, 148.8, 137.6, 137.4, 134.8,

(s, 1H), 8.83 (d, J = 3.5 Hz, 1H), 8.29 (d, J = 7.8 Hz, 1H),

6

C

O

https://dx.doi.org/10.1021/acsinfecdis.0c00511

ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

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Article

8

7

6

.00 (s, 1H), 7.80 (s, 1H), 7.57−7.44 (m, 2H), 7.34 (s, 2H),

(HU-4c). HU-4c was obtained as red solid (0.03 g, 65% yield).

1

.17 (s, 2H), 6.94 (s, 3H), 3.95 (d, J = 13.0 Hz, 4H), 3.80 (s,

H NMR (400 MHz, DMSO-d ) δ : 8.11 (d, J = 8.00 Hz, 1H),

6

H

13

H), 3.46 (s, 2H), 2.85 (s, 2H). C NMR (100 MHz, DMSO-

7.85−7.83 (m, 3H), 7.66−7.64 (m, 1H), 7.27−7.25 (d, J =

d ) δ : 166.6, 162.3, 153.6, 149.2, 149.1, 148.1, 147.5, 138.9,

6

C

8.00 Hz, 1H), 7.20 (s, 1H), 7.08−7.06 (d, J = 8.00 Hz, 1 H),

1

2

38.3, 137.2, 135.1, 133.7, 130.1, 129.7, 129.1, 126.7, 125.6,

24.4, 125.2, 124.8, 124.3, 122.9, 113.3, 112.4, 56.0, 55.9, 45.5,

3

.81 (s, 3H), 3.79 (s, 3 H), 3.64 (m, 4H), 3.47−3.44 (m, 8 H),

13

.23 (m, 4H), 2.05 (m, 2H). C NMR (100 MHz, CDCl ) δ :

3

C

8.6, 26.9. HRMS (ESI) calculated for C H N O S [M +

29

28

4

5

165.9, 160.5, 150.3, 149.0, 142.8, 141.9, 135.7, 135.1, 132.1,

+

H] m/z, 545.1859; found, 545.1855.

E)-3-(3,4-Dimethoxybenzylidene)-N-(2-(thiophene-3-

sulfonamido)ethyl)-2,3-dihydro-1H-cyclopenta[b]quinoline-

-carboxamide (HU-3c). HU-3c was obtained as yellow solid

130.8, 129.1, 128.4, 125.6, 124.7, 124.5, 124.2, 113.3, 112.3,

(

7

2.2, 70.8, 63.7, 60.4, 56.1, 55.9, 54.6, 51.7, 44.1, 36.6, 28.8,

+

27.1, 23.7. HRMS (ESI) calculated for C H N O [M + H]

2

9

33

3

4

9

m/z, 488.2549; found, 488.2556.

1

(

0.055 g, 75% yield). H NMR (400 MHz, DMSO-d ) δ :

6 H

(E)-3-(3,4-Dimethoxybenzylidene)-N-(3-(4-methylpipera-

zin-1-yl)propyl)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-

carboxamide (HU-4d). HU-4d was obtained as red solid (0.03

8

7

.76 (t, J = 5.6 Hz, 1H), 8.20 (d, J = 1.7 Hz, 1H), 8.01 (d, J =

.3 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.81−7.67 (m, 4H),

.52 (t, J = 7.2 Hz, 1H), 7.38 (dd, J = 5.1, 1.1 Hz, 1H), 7.25 (d,

1

g, 65% yield). H NMR (400 MHz, DMSO-d ) δ : 8.07−8.04

6

H

J = 11.1 Hz, 2H), 7.05 (d, J = 8.3 Hz, 1H), 3.85 (s, 3H), 3.81

(

m, 2H), 7.80−7.76 (m, 1H), 7.71 (s, 1H), 7.64−7.60 (m,

(

s, 3H), 3.44 (dd, J = 12.3, 6.3 Hz, 2H), 3.20 (d, J = 6.5 Hz,

H), 3.05−3.01 (m, 2H). C NMR (100 MHz, DMSO-d₆)

1

H), 7.21−7.18 (d, J = 8 Hz, 1H), 7.13 (s, 1H), 7.03−7.01 (d,

13

4

J = 8 Hz, 1H), 3.96 (m, 6H), 3.78−3.76 (m, 6H), 3.28−3.14

δ : 166.6, 162.4, 149.2, 149.2, 148.3, 140.8, 138.8, 138.5,

13

C

(

m, 4H), 1.94 (bs, 11H). C NMR (100 MHz, DMSO-d ) δ :

6 C

1

33.6, 130.9, 130.2, 129.7, 129.6, 129.3, 126.7, 125.1, 125.6,

24.9, 124.3, 122.9, 113.5, 112.4, 56.0, 55.9, 42.6, 28.6, 27.3,

1

6

67.6, 161.8, 149.9, 148.9, 144.4, 137.4, 136.8, 135.9, 131.4,

29.3, 129.2, 128.2, 125.9, 125.6, 124.1, 123.9, 113.1, 112.2,

+

2

6.8. HRMS (ESI) calculated for C H N O S [M + H] m/

28

27

3

5 2

6.7, 60.6, 56.6, 55.9, 55.8, 54.9, 21.3, 21.1, 18.4, 14.3. HRMS

z, 550.1470; found, 550.1479.

+

(

ESI) calculated for C H N O [M + H] m/z, 501.2866;

3

0

36

4

3

General Procedures for the Synthesis of Compounds HU-

found, 501.2824.

4

a−HU-6d. Triethyl amine (0.62 mmol) and T P (0.62

3

(

E)-N-(2-Aminoethyl)-3-(3,4-dimethoxybenzylidene)-2,3-

mmol) were added to acid intermediate 3 (0.20 mmol) and

amine reagents in dichloromethane (5 mL) at 0° C. The

reaction mixture was allowed to stir at RT overnight (approx.

dihydro-1H-cyclopenta[b]quinoline-9-carboxamide hydro-

chloride (HU-5). HU-5 was obtained as red solid (0.35 g,

1

6

(

7

8

3

3% yield). H NMR (400 MHz, DMSO-d ) δ : 8.15−8.13

6

H

1

0−12 h). The crude reaction mixture was diluted with

d, J = 8.00 Hz, 1H), 7.91−7.85 (m, 3H), 7.68−7.66 (t, 1H),

dichloromethane (15 mL), and water (20 mL) was added. The

organic layer was separated, washed with brine solution (20

mL), dried over anhydrous sodium sulfate, ﬁltered, and

concentrated using a rotary evaporator. The crude product

was then puriﬁed using silica gel (60−120 mesh) ﬂash column

chromatography. Gradient elution using 80%−100% EtOAc in

hexanes furnished the desired product.

.26−7.24 (d, J = 8.0 Hz, 1H), 7.16 (s, 1H), 7.06−7.04 (d, J =

.0 Hz, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 3.67−3.63 (t, 2H),

1

3

.22 (m, 4H), 3.10−3.07 (t, 2H). C NMR (100 MHz,

CDCl ) δ : 164.7, 159.9, 150.6, 148.9, 141.2, 138.1, 136.5,

3

C

1

33.9, 132.9, 132.7, 129.2, 128.8, 125.1, 124.9, 124.8, 123.7,

13.2, 112.2, 66.7, 55.9, 55.9, 42.9, 28.8, 26.8. HRMS (ESI)

+

calculated for C H ClN O [M + H] m/z, 404.1974; found,

2

4

26

3

(

E)-3-(3,4-Dimethoxybenzylidene)-N-(3-(pyrrolidin-1-yl)-

4

04.1963.

propyl)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-carboxa-

mide (HU-4a). HU-4a was obtained as red solid (0.03 g, 65%

yield). H NMR (400 MHz, DMSO-d ) δ : 8.08−8.06 (dt, J =

(

E)-(4,4-Diﬂuoropiperidin-1-yl)(3-(3,4-dimethoxybenzyli-

1

dene)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl) metha-

6

H

none (HU-6a). HU-6a was obtained as yellow solid (0.03 g,

1

3

0.9, 9.0 Hz, 1H), 7.84−7.60 (m, 4H), 7.20−7.03 (dt, J = 16.7,

0.3 Hz, 3H), 3.78 (m, 7H), 3.48 (dt, J = 13.6, 5.2 Hz, 4H),

.28−3.15 (m, 6H), 2.53−2.46 (m, 2H), 2.07−1.84 (m, 5H).

C NMR (100 MHz, DMSO-d ) δ : 168.0, 149.5, 149.2,

1

6

5% yield). H NMR (400 MHz, CDCl ) δ : 8.13 (d, J = 8.5

3

H

Hz, 1H), 7.80 (s, 1H), 7.74−7.60 (m, 3H), 7.58−7.47 (m,

1

3

1H), 7.17 (s, 1H), 7.02−6.89 (m, 1H), 3.95 (s, 6H), 3.33 (d, J

6

C

1

3

=

9.3 Hz, 4H), 2.32−2.12 (bs, 4H), 1.92−1.85 (bs, 4H). C

1

36.7, 129.9, 129.8, 127.4, 125.6, 124.4, 124.1, 123.3, 113.5,

12.4, 56.1, 55.9, 53.4, 52.2, 36.7, 28.6, 28.4, 27.0, 26.1, 13.1.

NMR (100 MHz, CDCl ) δ : 166.6, 162.5, 149.0, 148.8,

3

C

+

137.5, 136.7, 132.9, 130.2, 129.9, 129.3, 127.3, 125.9, 124.6,

HRMS (ESI) calculated for C H N O [M + H] m/z,

29

33

3

1

2

23.7, 122.8, 121.1, 113.4, 112.9, 56.2, 55.9, 43.4, 38.2, 34.0,

8.6, 26.7. HRMS (ESI) calculated for C H F N O [M +

4

72.2600; found, 472.2583.

E)-3-(3,4-Dimethoxybenzylidene)-N-(3-(piperidin-1-yl)-

(

27 26

2

3

+

H] m/z, 465.1990; found, 465.1997.

propyl)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-carboxa-

mide (HU-4b). HU-4b was obtained as red solid (0.03 g, 65%

yield). H NMR (400 MHz, DMSO-d ) δ : 8.06−8.04 (dt, J =

(

E)-(3-(3,4-Dimethoxybenzylidene)-2,3-dihydro-1H-

1

cyclopenta[b]quinolin-9-yl)(4-(triﬂuoromethyl)piperidin-1-

6

H

yl) methanone (HU-6b). HU-6b was obtained as red solid

1

3

5.9, 8.0 Hz, 1H), 7.80−7.58 (m, 4H), 7.23−7.03 (m, 3H),

.78−3.75 (m, 6H), 3.63 (s, 1H), 3.53 (s, 2H), 3.43 (s, 3H),

.25−3.07 (bs, 6H), 2.88−2.82 (m, 2H), 1.99 (s, 1H), 1.81 (d,

1

(

0.03 g, 65% yield). H NMR (400 MHz, DMSO-d ) δ : 8.04

6

H

d, J = 8.2 Hz, 1H), 7.77−7.66 (m, 2H), 7.62−7.52 (m, 2H),

13

J = 12.5 Hz, 2H), 1.66 (s, 3H). C NMR (100 MHz, DMSO-

d ) δ : 166.8, 161.8, 149.5, 148.9, 146.3, 139.6, 137.3, 134.4,

7.25 (d, J = 12.3 Hz, 2H), 7.06 (d, J = 8.1 Hz, 1H), 4.79 (s,

1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.19−3.13 (m, 7H), 2.65 (m,

1H), 2.03−2.00 (d, J = 14.0 Hz, 1H), 1.75−1.58 (m, 1H),

6

C

1

30.7, 129.7, 127.9, 126.8, 127.1, 126.8, 125.4, 123.9, 113.1,

1

3

1

12.3, 66.7, 55.9, 55.9, 55.9, 54.4, 52.9, 36.7, 24.0, 22.9, 21.5.

1.16−1.18 (m, 2H). C NMR (100 MHz, DMSO-d ) δ :

6 C

+

HRMS (ESI) calculated for C H N O [M + H] m/z,

165.6, 165.5, 162.6, 162.4, 162.1, 149.2, 149.2, 148.3, 148.1,

138.5, 138.3, 137.9, 137.8, 133.5, 133.0, 130.2, 130.1, 129.9,

129.8, 129.6, 129.2, 127.1, 125.2, 125.1, 124.7, 124.0, 123.7,

122.9, 122.9, 113.5, 113.3, 112.4, 56.0, 55.9, 55.9, 45.1, 44.8,

30

35

3

38.2232; found, 338.2198.

E)-3-(3,4-Dimethoxybenzylidene)-N-(3-morpholinoprop-

yl)-2,3-dihydro-1H-cyclopenta[b]quinoline-9-carboxamide

(

P

https://dx.doi.org/10.1021/acsinfecdis.0c00511

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1

2

8.5, 28.5, 26.6, 26.5, 25.7, 25.5, 24.6. HRMS (ESI) calculated

g, 63% yield). H NMR (400 MHz, DMSO-d ) δ : 8.20 (d, J =

6

H

+

for C H F N O [M + H] m/z, 497.2052; found, 497.2059.

8.5 Hz, 1H), 7.94 (s, 1H), 7.92−7.87 (m, 1H), 7.82 (d, J = 8.2

Hz, 1H), 7.73−7.68 (m, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.19 (s,

1H), 7.07 (d, J = 8.5 Hz, 1H), 3.80 (d, J = 9.3 Hz, 6H), 3.70−

3.62 (m, 2H), 3.51−3.46 (m, 2H), 3.46−3.41 (m, 2H), 3.33−

2

8

27

3

2

3

(

E)-(4-Aminopiperidin-1-yl)(3-(3,4-dimethoxybenzyli-

dene)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl) metha-

none (HU-6c). HU-6c was obtained as red solid (0.03 g,

1

13

6

5% yield). H NMR (400 MHz, DMSO-d ) δ : 8.20−8.10

3.21 (m, 4H), 3.20−3.10 (m, 2H). C NMR (100 MHz,

6

H

(m, 1H), 7.93−7.88 (m, 1H), 7.81−7.75 (m, 1H), 7.71−7.65

(m, 2H), 7.28−7.18 (m, 2H), 7.10−7.04 (m, 1H), 4.69−4.66

(t, 1H), 3.80 (s, 3H), 3.79 (s, 3H), 3.32−3.23 (m, 6H), 3.19−

DMSO-d ) δ : 164.7, 160.2, 150.6, 149.1, 141.3, 141.1, 136.4,

6

C

134.1, 132.8, 132.5, 129.1, 128.9, 125.2, 124.8, 123.8, 113.3,

112.3, 72.2, 70.8, 60.4, 56.0, 55.9, 28.8. HRMS (ESI)

calculated for C H N O [M + H] m/z, 430.2131; found,

+

3

1

2

.04 (m, 2H), 2.21−2.09 (m, 1H), 1.79−1.70 (m, 1H), 1.56−

2

6

27

3

1

3

.40 (m, 2H). C NMR (100 MHz, DMSO-d ) δ : 168.6,

430.2129.

6

C

67.9, 162.2, 162.1, 149.5, 149.2, 149.2, 141.4, 137.5, 136.7,

30.9, 130.1, 129.9, 129.7, 128.4, 127.4, 125.6, 124.1, 123.8,

23.6, 123.3, 113.4, 112.4, 58.6, 56.1, 47.9, 44.2, 41.7, 37.9,

9.7, 28.6, 26.9, 21.7. HRMS (ESI) calculated for C H N O

(E)-(4-(Cyclopropanecarbonyl)piperazin-1-yl)(3-(3,4-di-

methoxybenzylidene)-2,3-dihydro-1H-cyclopenta[b]-

quinolin-9-yl) methanone (HU-7b). HU-7b was obtained as

1

yellow solid (0.05 g, 65% yield). H NMR (400 MHz, CDCl )

2

7

29

3

+

[

M + H] m/z, 444.2287; found, 444.2245.

E)-N-(1-(3-(3,4-Dimethoxybenzylidene)-2,3-dihydro-1H-

δ_H: 8.14 (d, J = 8.4 Hz, 1H), 7.81 (s, 1H), 7.68 (dd, J = 14.2,

7.0 Hz, 2H), 7.54−7.46 (m, 1H), 7.22 (s, 1H), 7.16 (s, 1H),

6.94 (d, J = 8.4 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.63−3.56

(m, 4H), 3.34−3.24 (m, 4H), 3.13 (dd, J = 14.6, 7.3 Hz, 4H),

3.06−2.98 (m, 1H), 1.26 (d, J = 9.1 Hz, 1H), 1.01 (s, 2H),

(

cyclopenta[b]quinoline-9-carbonyl)piperidin-4-yl)-2-naph-

thamide (HU-6d). HU-6d was obtained as yellow solid (0.03

g, 65% yield). H NMR (400 MHz, CDCl ) δ : 8.26 (s, 1H),

1

3

H

1

3

8

.14−8.11 (d, 1H), 7.93−7.86 (m, 3H), 7.81−7.77 (m, 2H),

0.81 (m, 2H). C NMR (100 MHz, CDCl ) δ : 171.8, 168.3,

3 C

7

.68−7.50 (m, 4H), 7.42 (s, 1H), 6.95−6.96 (m, 1H), 6.24

165.8, 162.6, 149.2, 148.6, 138.4, 137.6, 130.1, 129.9, 129.6,

127.2, 125.6, 125.1, 124.9, 122.9, 113.5, 112.4, 56.0, 55.9, 46.0,

28.6, 28.3, 26.5, 10.8, 9.0, 7.6. HRMS (ESI) calculated for

(

m, 1H), 5.09 (m, 1H), 3.95−3.90 (s, 6H), 3.28−3.14 (m,

13

7

H), 2.16−2.13 (m, 1H), 1.38−1.35 (m, 4H). C NMR (100

+

MHz, CDCl ) δ : 166.9, 166.4, 162.1, 148.9, 137.7, 134.8,

C H N O [M + H] m/z, 498.2393; found, 498.2399.

3

C

30 31

3

4

1

32.8, 132.6, 131.4, 130.3, 129.7, 129.5, 129.4, 128.9, 128.6,

27.8, 127.4, 126.9, 126.8, 126.5, 124.0, 123.4, 122.7, 122.6,

(E)-(3-(3,4-Dimethoxybenzylidene)-2,3-dihydro-1H-

cyclopenta[b]quinolin-9-yl)(4-(thiophene-2-carbonyl)-

piperazin-1-yl)methanone (HU-7c). HU-7c was obtained as

12.9, 111.2, 55.9, 55.9, 47.3, 45.4, 40.6, 37.9, 32.4, 28.7.

+

1

HRMS (ESI) calculated for C H N O [M + H] m/z,

yellow solid (0.045 g, 55% yield). H NMR (400 MHz,

38

35

3

4

5

98.2706; found, 598.2655.

General Procedures for the Synthesis of Compounds HU-

a to HU-7e. Synthesis of HU-7a: Triethyl amine (0.42 g, 4.1

CDCl ) δ : 8.32 (bs, 1H), 8.12 (bs, 1H), 7.71−7.66 (m, 2H),

3

H

7.54−7.45 (m, 2H), 7.29−7.23 (m, 2H), 7.16 (s, 1H), 7.04−

7.02 (m, 1H), 6.93−6.91 (d, 1H), 4.13−4.08 (m, 4H), 3.99−

3.88 (s, 6H), 3.77−3.71 (m, 2H), 3.37−3.35 (m, 4H), 3.15

7

mmol) and T P (1.32 g, 4.1 mmol) were added to acid 3 (0.5

3

1

3

g, 1.38 mmol) in dichloromethane (5 mL) at 0° C. The

reaction mixture was allowed to stir at RT for 15 min before

the addition of Boc-protected amine (0.26 g, 1.66 mmol), and

stirring was continued overnight (approx. 14−16 h).

(m, 2H). C NMR (100 MHz, CDCl ) δ : 165.8, 165.3,

3 C

162.5, 149.2, 149.2, 148.2, 138.4, 137.6, 136.3, 133.4, 130.1,

129.9, 129.6, 127.8, 127.7, 127.1, 125.1, 124.9, 123.9, 122.8,

113.5, 112.4, 56.0, 55.9, 46.7, 46.9, 28.6, 26.5. HRMS (ESI)

+

Synthesis of HU-7b−HU-7d: Triethyl amine (0.62 mmol)

calculated for C H N O S [M + H] m/z, 540.1870; found,

3

1

29

3

4

and T P (0.62 mmol) were added to a stirred mixture of acids

540.1868.

3

and HU-7a (0.24 mmol) in dichloromethane (2 mL) at 0° C.

The reaction contents were allowed to stir at RT overnight

(E)-(3-(3,4-Dimethoxybenzylidene)-2,3-dihydro-1H-

cyclopenta[b]quinolin-9-yl)(4-(thiophene-3-carbonyl)-

(

approx. 10−12 h). The crude reaction mixture was diluted

piperazin-1-yl)methanone (HU-7d). HU-7d was obtained as

1

with dichloromethane (15 mL), and water (20 mL) was added.

The organic layer was separated, washed with brine solution

(

concentrated using a rotary evaporator. The crude product was

then puriﬁed using silica gel (60−120 mesh) ﬂash column

chromatography. Gradient elution using 100% EtOAc

furnished the desired product.

yellow solid (0.05 g, 75% yield H NMR (400 MHz, CDCl )

3

δ : 8.12 (d, J = 8.3 Hz, 1H), 7.79 (s, 1H), 7.72−7.64 (m, 2H),

H

20 mL), dried over anhydrous sodium sulfate, ﬁltered, and

7.51 (dd, J = 15.3, 7.5 Hz, 2H), 7.35 (s, 1H), 7.24 (d, J = 8.4

Hz, 1H), 7.17 (s, 2H), 6.95 (d, J = 8.4 Hz, 1H), 3.95 (s, 3H),

3.94 (s, 3H), 3.87 (d, J = 29.3 Hz, 2H), 3.49 (s, 4H), 3.32

(ddd, J = 21.6, 13.8, 7.1 Hz, 4H), 3.05 (ddd, J = 18.3, 12.3, 7.1

1

3

Hz, 2H). C NMR (100 MHz, CDCl ) δ : 165.8, 165.3,

3

C

Synthesis of HU-7e: Triethyl amine (0.62 mmol) and

sulfonyl chloride (0.62 mmol) were added to HU-7a (0.20

mmol) in dichloromethane (2 mL) at 0° C. The reaction

mixture was allowed to stir at RT for 3 h. The crude reaction

mixture was diluted with dichloromethane (15 mL), and water

162.5, 149.2, 149.2, 148.2, 138.4, 137.6, 136.3, 133.4, 130.1,

129.9, 129.6, 127.8, 127.7, 127.1, 125.1, 124.9, 123.9, 122.8,

113.5, 112.4, 56.0, 55.9, 46.7, 46.9, 28.6, 26.5. HRMS (ESI)

+

calculated for C H N O S [M + H] m/z, 540.1957; found,

3

1

29

3

4

540.1951.

(

20 mL) was added. The organic layer was separated, washed

(E)-(3-(3,4-Dimethoxybenzylidene)-2,3-dihydro-1H-

cyclopenta[b]quinolin-9-yl)(4-(pyridin-3-ylsulfonyl)-

piperazin-1-yl) methanone (HU-7e). HU-7e was obtained as

with brine solution (20 mL), dried over anhydrous sodium

sulfate, ﬁltered, and concentrated using a rotary evaporator.

The crude product was puriﬁed using silica gel (60−120 mesh)

ﬂash column chromatography using gradient elution with

1

red solid (0.03 g, 65% yield). H NMR (400 MHz, CDCl ) δ :

3

H

8.97 (d, J = 1.8 Hz, 1H), 8.88 (dd, J = 4.8, 1.5 Hz, 1H), 8.11

(d, J = 8.4 Hz, 1H), 8.08−8.01 (m, 1H), 7.78 (s, 1H), 7.66 (t, J

= 7.6 Hz, 1H), 7.54 (dd, J = 8.0, 4.8 Hz, 2H), 7.48 (d, J = 7.5

Hz, 1H), 7.43−7.36 (m, 1H), 7.15 (s, 1H), 6.94 (d, J = 8.4 Hz,

1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.57 (d, J = 5.7 Hz, 4H),

5

0%−100% EtOAc in hexane to obtain the pure products.

(

E)-(3-(3,4-Dimethoxybenzylidene)-2,3-dihydro-1H-

cyclopenta[b]quinolin-9-yl)(piperazin-1-yl)methanone hy-

drochloride (HU-7a). HU-7a was obtained as red solid (0.35

Q

https://dx.doi.org/10.1021/acsinfecdis.0c00511

ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

pubs.acs.org/journal/aidcbc

Article

1

3

+

3

(

.38−3.27 (m, 4H), 3.21 (dd, J = 16.6, 8.4 Hz, 4H). C NMR

calculated for C H N O [M + H] m/z, 467.2811; found,

30 34 4

467.2755.

100 MHz, CDCl ) δ : 165.8, 165.3, 149.2, 149.2, 148.2,

3

C

1

2

38.4, 137.6, 136.3, 133.4, 130.1, 129.8, 129.6, 127.8, 127.7,

27.1, 125.1, 124.9, 123.9, 122.8, 113.5, 112.4, 56.0, 55.9, 47.2,

(E)-(4-(Cyclopropanecarbonyl)piperazin-1-yl)(3-(4-

(dimethylamino)benzylidene)-2,3-dihydro-1H-cyclopenta-

[b]quinolin-9-yl)methanone (HU-8e). HU-8e was obtained as

8.6, 26.5, 16.9, 15.7. HRMS (ESI) calculated for

+

1

C H N O S [M + H] m/z, 571.2015; found, 571.2010.

brown red solid (0.03 g, 65% yield). H NMR (400 MHz,

3

1

30

4

5

General Procedures for the Synthesis of Compounds HU-

a−HU-9f. HU-8a-HU-8e were synthesized as HU-2a-HU-7d

DMS0-d ) δ : 8.10−8.79 (m, 2H), 7.88−7.69 (m, 2H), 7.65−

6 H

8

7.63 (m, 1H), 7.52−7.45 (m, 2H), 6.80−6.71 (m, 2H), 3.64−

3.50 (m, 3H), 3.24−3.12 (m, 4H), 3.06−3.00 (m, 2H), 2.95

with immediate 4 or 7 as the substrate. Compounds HU-9a-

HU-9f were synthesized from immediate 2.

(

s, 6H), 1.51−1.47 (m, 2H), 1.16−1.06 (m, 2H), 0.91−0.88

13

m, 1H), 0.76−0.72 (m, 2H). C NMR (100 MHz, DMSO)

(

E)-3-(4-(Dimethylamino)benzylidene)-N-(2-(furan-3-

δ : 182.8, 171.8, 165.9, 162.9, 162.1, 150.3, 148.3, 137.3,

carboxamido)ethyl)-2,3-dihydro-1H-cyclopenta[b]-

quinoline-9-carboxamide (HU-8a). HU-8a was obtained as

brown solid (0.025 g, 41% yield). H NMR (400 MHz,

DMSO-d ) δ : 7.97 (s, 1H), 7.90−7.88 (m, 2H), 7.66−7.64

C

1

35.4, 133.4, 131.3, 129.7, 129.4, 126.7, 125.6, 125.0, 124.8,

1

23.7, 112.6, 48.2, 37.9, 28.7, 26.5, 10.8, 7.6. HRMS (ESI)

+

calculated for C H N O [M + H] m/z, 481.2604; found,

30 32

4

2

6

H

4

81.2605.

N-(4-(Dimethylamino)butyl)-2,3-dihydro-1H-cyclopenta-

b]quinoline-9-carboxamide (HU-9a). HU-9a was obtained

(

1

4

m, 4H), 7.57−7.47 (m, 2H), 7.26−7.21 (m, 2H), 6.74−6.67

13

m, 1H), 3.79 −3.74 (m, 2H), 3.21−2.71 (m, 12H). C NMR

100 MHz, DMSO-d ) δ : 163.4, 162.1, 150.1, 149.9, 145.2,

[

6

C

1

as white solid (0.056 g, 87% yield). H NMR (400 MHz,

DMSO-d ) δ : 8.02 (d, J = 8.3 Hz, 1H), 7.96−7.82 (m, 2H),

7

43.7, 133.9, 131.4, 131.2, 129.5, 129.1, 127.8, 127.4, 126.7,

26.5, 126.1, 124.6, 123.9, 123.5, 122.5, 112.0, 108.6, 45.3,

0.8, 40.2, 40.2, 39.8. HRMS (ESI) calculated for C H N O

3

6

H

.71 (d, J = 7.6 Hz, 1H), 3.39 (d, J = 6.6 Hz, 2H), 3.34−3.19

2

9

28

4

+

(m, 2H), 3.18−3.01 (m, 4H), 2.74 (s, 6H), 2.22 (d, J = 7.3 Hz,

2

[

M + H] m/z, 481.2240; found, 481.2031.

1

3

H), 1.65 (d, J = 33.9 Hz, 4H). C NMR (100 MHz, CDCl )

3

(

E)-(4,4-Diﬂuoropiperidin-1-yl)(3-(4-(dimethylamino)-

δ_C: 167.1, 164.9, 143.9, 139.4, 135.4, 132.7, 129.3, 126.1,

24.2, 122.6, 56.7, 42.6, 32.8, 29.5, 26.3, 23.4, 23.3, 21.8.

benzylidene)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-yl)-

methanone (HU-8b). HU-8b was obtained as brown solid

1

+

1

HRMS (ESI) calculated for C19

12.2017; found, 312.2044.

N-(3-(Piperidin-1-yl)propyl)-2,3-dihydro-1H-cyclopenta-

b]quinoline-9-carboxamide (HU-9b). HU-9b was obtained

H N O [M + H] m/z,

25 3

(

0.03 g, 65% yield). H NMR (400 MHz, DMSO) δ : 8.11 (d,

H

3

J = 8.5 Hz, 1H), 7.94 (s, 1H), 7.88 (t, J = 7.6 Hz, 1H), 7.75 (d,

J = 8.1 Hz, 1H), 7.70−7.63 (m, 1H), 7.51 (d, J = 8.9 Hz, 2H),

[

6

1

(

.77 (d, J = 8.9 Hz, 2H), 4.04−3.95 (m, 1H), 3.86−3.74 (m,

1

as white solid (0.045 g, 75% yield). H NMR (400 MHz,

DMSO-d ) δ : 8.16 (d, J = 8.3 Hz, 1H), 7.98 (t, J = 9.2 Hz,

H), 7.82 (t, J = 7.6 Hz, 1H), 3.41 (dt, J = 15.7, 7.1 Hz, 6H),

H), 3.53 (s, 6H), 3.27 (dd, J = 18.4, 11.6 Hz, 2H), 3.09−3.02

_m_,₂_H₎_,₂_.₉₁₍_s_,₆_H₎_.1 C NMR (100 MHz, CDCl ) δ :

3

6

H

3

C

2

3

2

1

6

73.5, 164.1, 159.0, 151.1, 142.4, 138.3, 137.2, 136.5, 133.6,

33.5, 129.0, 128.3, 125.3, 124.2, 123.2, 122.6, 120.8, 113.2,

6.7, 48.9, 46.4, 34.2, 33.2, 29.1, 26.7, 21.3, 18.4. HRMS (ESI)

.15−3.05 (m, 4H), 2.87 (t, J = 11.4 Hz, 2H), 2.33−2.22 (m,

H), 2.09−1.97 (m, 2H), 1.80 (d, J = 11.3 Hz, 2H), 1.76−1.63

13

(

m, 3H), 1.42 (dd, J = 27.2, 13.9 Hz, 1H). C NMR (100

+

calculated for C H F N O [M + H] m/z, 498.2200; found,

2

7

27

2

3

MHz, DMSO-d ) δ : 167.2, 165.1, 143.5, 139.5, 135.4, 132.7,

6

C

4

48.2185.

E)-(3-(4-(Dimethylamino)benzylidene)-2,3-dihydro-1H-

cyclopenta[b]quinolin-9-yl)(4-(2,2,2-triﬂuoroethyl)piperazin-

-yl)methanone (HU-8c). HU-8c was obtained as dark brown

1

2

29.3, 126.2, 124.2, 122.7, 54.3, 52.6, 36.8, 32.8, 29.5, 23.9,

3.4, 22.9, 21.6. HRMS (ESI) calculated for C H N O [M +

(

2

1

27

3

+

H] m/z, 338.2232; found, 338.2198.

1

N-(3-(4-Methylpiperazin-1-yl)propyl)-2,3-dihydro-1H-

1

solid (0.03 g, 65% yield). H NMR (400 MHz, DMSO-d ) δ :

6

H

cyclopenta[b]quinoline-9-carboxamide (HU-9c). HU-9c was

8

.15−8.13 (d, J = 8 Hz, 1H), 8.06 (s, 1H), 7.94−7.92 (m, 1H),

1

obtained as white solid (0.035 g, 65% yield). H NMR (400

7

.73−7.71 (m, 2H), 7.60−7.58 (d, J = 8 Hz, 2H), 6.90−6.88

MHz, DMSO-d ) δ : 8.65 (d, J = 5.1 Hz, 1H), 7.95 (d, J = 8.2

6

H

(

d, J = 8 Hz, 2H), 3.38 (m, 8H), 3.24−3.14 (m, 5H), 3.07−

13

Hz, 1H), 7.81 (d, J = 7.8 Hz, 1H), 7.68 (t, J = 7.0 Hz, 1H),

3

.02 (m, 3H), 2.83−2.79 (m, 4H). C NMR (100 MHz,

7

.54 (t, J = 7.5 Hz, 1H), 3.23−2.99 (m, 6H), 2.45−2.32 (m,

DMSO-d ) δ : 165.6, 162.9, 150.3, 148.3, 137.4, 135.3, 133.2,

6

C

8H), 2.23 (s, 3H), 2.19−2.08 (m, 3H), 1.74 (dd, J = 13.9, 7.0

Hz, 3H). C NMR (100 MHz, DMSO-d ) δ : 168.1, 166.4,

1

31.2, 129.7, 129.4, 127.8, 126.6, 125.6, 125.0, 124.7, 123.7,

13

6

C

12.6, 57.1, 56.8, 53.9, 53.3, 46.3, 41.3, 28.7, 26.5. HRMS

1

47.6, 141.6, 141.4, 138.3, 131.9, 129.1, 129.0, 126.5, 125.3,

+

(ESI) calculated for C H F N O [M + H] m/z, 495.2372;

28

29

3

4

123.7, 55.6, 54.7, 53.8, 52.5, 34.5, 29.5, 26.7, 26.5, 23.4. HRMS

+

found, 495.2344.

(

ESI) calculated for C H N O [M + H] m/z, 353.2341;

21 28 4

(

E)-(4-(Cyclopropylmethyl)piperazin-1-yl)(3-(4-

found, 353.2310.

(

[

dimethylamino)benzylidene)-2,3-dihydro-1H-cyclopenta-

b]quinolin-9-yl)methanone (HU-8d). HU-8d was obtained

N-(3-Morpholinopropyl)-2,3-dihydro-1H-cyclopenta[b]-

quinoline-9-carboxamide (HU-9d). HU-9d was obtained as

1

as red solid (0.03 g, 65% yield). H NMR (400 MHz, DMSO-

d ) δ : 8.10 (d, J = 8.7 Hz, 1H), 7.77 (s, 1H), 7.64 (dd, J = 7.9,

6

white solid (0.03 g, 65% yield). H NMR (400 MHz, DMSO-

H

d ) δ : 8.03 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 8.2 Hz, 1H), 7.64

6

H

6

2

1

.0 Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H), 7.48−7.41 (m, 1H),

.76 (d, J = 8.8 Hz, 2H), 3.35−3.20 (m, 4H), 3.02 (s, 6H),

.75 (m, 2H), 2.41 (s, 3H), 1.31 (dd, J = 20.2, 12.3 Hz, 3H),

.00 (dd, J = 15.3, 7.6 Hz, 3H), 0.87 (ddd, J = 11.7, 7.6, 2.9

(t, J = 7.6 Hz, 1H), 7.48 (t, J = 7.6 Hz, 1H), 3.68 (dd, J = 11.8,

5.9 Hz, 2H), 3.31 (s, 4H), 3.14 (dt, J = 23.3, 7.5 Hz, 4H), 2.54

(t, J = 6.2 Hz, 2H), 2.39 (s, 4H), 2.21 (p, J = 7.5 Hz, 2H), 1.85

1

3

(dd, J = 12.4, 6.2 Hz, 2H). C NMR (100 MHz, DMSO-d₆)

δ_C: 167.9, 166.9, 147.8, 137.5, 131.9, 129.2, 128.8, 126.3,

124.5, 123.3, 66.6, 57.9, 53.6, 39.8, 34.7, 29.7, 24.7, 23.4.

13

Hz, 3H), 0.57 (s, 1H). C NMR (100 MHz, DMSO) δ_C:

1

4

65.5, 162.9, 150.3, 148.3, 137.6, 135.3, 133.1, 131.2, 129.7,

29.4, 126.6, 125.6, 125.0, 124.7, 123.7, 112.6, 62.9, 53.8, 52.9,

6.4, 41.4, 40.6, 28.7, 26.5, 8.6, 4.1, 4.1. HRMS (ESI)

+

HRMS (ESI) calculated for C H N O [M + H] m/z,

2

0

25

3

2

340.2025; found, 340.1998.

R

https://dx.doi.org/10.1021/acsinfecdis.0c00511

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ACS Infectious Diseases

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Article

(

4,4-Diﬂuoropiperidin-1-yl)(2,3-dihydro-1H-cyclopenta-

Lysates were transferred into 96-well plates, and luciferase

activity was measured using Glo-Max plate reader (Promega).

[

b]quinolin-9-yl)methanone (HU-9e). HU-9e was obtained as

white solid (0.04 g, 90% yield). H NMR (400 MHz, CDCl )

1

5

Cell Viability Assays. CEM T cells (2 × 10 ) were grown

3

δ : 8.74 (d, J = 8.5 Hz, 1H), 7.97−7.90 (m, 1H), 7.84 −7.76

in 96-well plates and treated overnight with serially diluted

compounds or DMSO control. To assess cytotoxicity with

MTT, 10 μL of MTT solution (ATCC 30-1010KTM) was

added to each well, and the samples were incubated at 37 °C

for 2 h. Then 100 μL of lysis buﬀer (0.03% HCl with 10%

SDS) was added to each well. The cells were incubated for 20

min in the dark. Absorbance was measured at 490 nm at a

microplate reader (Bio-Rad Model 680, United States). Each

point was measured in triplicate, and serum-free medium with

MTT solution was used as a negative control.

Assays to Measure HIV-1 IIIB Virus Replication. CEM

T cells and peripheral blood mononuclear cells (PBMCs) were

infected with HIV-1 IIIB virus. Infected cells were treated for

an additional 72 h with 10 μM of compounds HU-1a, HU-1c,

HU-1d, and 1E7-03 and 25 μM azidothymidine (AZT).

Control cells were untreated or treated with DMSO. For

quantitative analysis of HIV-1 gag mRNA expression, total

RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad,

CA) according to the manufacturer’s protocol. Total RNA

H

(

m, 2H), 4.16 (dd, J = 12.9, 6.7 Hz, 1H), 4.05- 3.97 (m, 1H),

3

2

.80−3.61 (m, 2H), 3.38−3.23 (m, 3H), 3.09−2.96 (m, 1H),

.51−2.39 (m, 2H), 2.20 (dt, J = 17.3, 6.0 Hz, 2H), 1.96−1.84

1

3

(

m, 2H). C NMR (100 MHz, CDCl ) δ : 165.4, 163.8,

3 C

1

3

33.1, 129.8, 124.5, 124.1, 120.6, 43.4, 38.6, 37.9, 35.1, 34.9,

4.6, 34.1, 33.9, 33.7, 32.4, 29.2, 23.6. HRMS (ESI) calculated

+

for C H F N O [M + H] m/z, 317.1465; found, 317.1439.

1

8

18

2

(

2,3-Dihydro-1H-cyclopenta[b]quinolin-9-yl)(4-(2-

(

dimethylamino)ethyl)piperazin-1-yl)methanone (HU-9f).

HU-9f was obtained as white solid (0.05 g, 83% yield). H

NMR (400 MHz, DMSO-d ) δ : 8.16 (d, J = 8.5 Hz, 1H),

1

6

H

8

6

1

.05−7.96 (m, 2H), 7.86 (t, J = 7.6 Hz, 1H), 3.55−3.49 (m,

H), 3.41−3.36 (m, 4H), 2.82 (s, 4H), 2.77 (s, 4H), 1.51−

13

.43 (m, 4H), 0.94−0.87 (m, 2H). C NMR (100 MHz,

DMSO-d ) δ : 167.3, 164.1, 142.5, 138.2, 136.3, 133.5, 130.2,

6

C

1

4

25.7, 123.8, 122.0, 52.3, 52.0, 51.6, 51.5, 50.9, 50.8, 50.2, 49.0,

3.2, 43.0, 42.2. HRMS (ESI) calculated for C H N O [M +

21

28

4

+

H] m/z, 353.2341; found, 353.2255.

(

100 ng) was reverse transcribed to cDNA using Superscript

RT-PCR kit (Invitrogen, Carlsbad, CA). Hexamers and oligo-

dT were used as primers. For real-time PCR analysis, cDNA

was ampliﬁed using a Roche LightCycler 480 (Roche

Diagnostics) and SYBR Green 1 Master mix (Roche

Diagnostics). PCR was run for 45 cycles, and each cycle

included denaturation at 95 °C for 10 seconds, annealing at 60

METHODS

■

Chemicals and Cells. 1E7-03 analogues were synthesized

as described above. Their structures were veriﬁed by proton

and carbon nuclear magnetic resonance ( H NMR and

1

13

C

NMR), and their composition was determined to be ≥95%

pure by nano LC-FT/MS analysis (Figure S2). Compounds

were dissolved in DMSO to obtain 10 mM stock solutions.

Acetonitrile and water containing 0.1% formic acid (FA) were

Optima LC/MS grade (Fisher Scientiﬁc, Fair Lawn, NJ). High-

purity nitrogen (99.9%) was purchased from Roberts Oxygen

Co, Inc. (Rockville, MD). Other reagents were of analytical

grade. Dimethyl sulfoxide (DMSO) and acetone were from

Fisher Scientiﬁc (Fair Lawn, NJ). CD4+ T cells (CEM T)

were purchased from the American Type Culture Collection

Manassas, VA). The CEM T cells sand PBMCs were cultured

in RPMI medium (Invitrogen) containing 10% FBS and 1%

antibiotic solution (penicillin and streptomycin).

HIV-1 Inhibitory Assay Using VSV-G Pseudotyped

Virus. CEM T cells (6 × 10 cells/ml) were infected with

VSVG-pseudotyped pNL4-3.Luc.R-E-virus (HIV-1-LUC-G)

°

C for 10 seconds, and extension at 72 °C for 10 seconds.

Quantiﬁcation of gag was carried out using 18S RNA as a

normalization standard. The following primer sequences were

used: gag, forward- ATAATCCACCTATCCCAGTAGGAGA-

AAT, reverse- TITGGTCCITGTCITATGTCCAGAATGC

and 18SrRNA, forward- GCTGTTGCTACATCGACCTTT,

reverse- CTCCAGGTTTTGCAACCAGT. HIV capsid p24

protein was quantiﬁed by Alliance HIV-1 p24 ELISA Kit

(PerkinElmer, Waltham, MA) according to the manufacturer’s

instructions. A quantiﬁcation range of 12.5−100 pg/mL was

utilized in the present study. Linear regression ﬁtting was used

to estimate the concentration of p24. At least 3 biological

replicates were performed for each condition (culture

supernatants were collected from drug-treated CEM T cells).

LC/FT-MS Analysis of Small Molecules. An aliquot of 10

μL sample was loaded onto a nano HPLC system (LC-20AD,

Shimadzu Corporation, Columbia, MD) coupled to a LTQ XL

Orbitrap mass spectrometer (Thermo Fisher Scientiﬁc) with

the installed Xcalibur software (version 2.0.7, Thermo Fisher

Scientiﬁc). Liquid chromatography was carried out on a nano-

HPLC column packed with reverse phase PolySulfoethyl A, 5

μM, 200 Å (PolyLC Inc., Columbia, MD). Mobile phase A was

0.1% formic acid in water, and mobile phase B was 0.1% formic

acid in acetonitrile. The elution was performed at a ﬂow rate of

600 nL/min over 40 min using a multi-segment linear gradient

of mobile phase B as follows: 0−6.02 min, 1% B; 6.02−6.11

min, 1%−2% B; 6.11−20 min, 2%−80% B; 20−25 min, 80% B;

25−30 min, 80%−85% B; 30−31 min, 80%−2% B; 31−40

min, 2% B (v/v). The Orbitrap was operated under data-

dependent acquisition mode. The spray voltage, capillary

temperature, and capillary voltage were set to 2.0 kV, 200 °C,

and 39.5 V, respectively. Full-scan mass spectra were acquired

in Orbitrap over 150−1500 m/z with a resolution of 30 000,

(

5

17

overnight as previously described, seeded in 96-well white

5

plates (2 × 10 cells/ml, 90 μL/well) and cultured in complete

RPMI media at 37 °C in the presence of 5% CO . The cells

2

were treated with 3, 10, and 30 μM compounds for the

screening. For detailed analysis, compounds were serially

diluted using RPMI media as a vehicle control. At 24 h post-

treatment, 100 μL of reconstituted luciferase buﬀer (Luclite

Kit, Perkin Elmer) was added to each well; the samples were

incubated for 10 min, and luminescence was measured using a

Glo-Max Microplate Multimode reader (Promega).

HIV-1 Transcription Inhibitory Assay. 293T cells were

cultured in 24-well plates in DMEM media to reach 30%

conﬂuence. The cells were transfected with plasmids expressing

HIV-1 LTR Luciferase reporter, Flag-tagged Tat protein, and

PP1γ-EGFP as indicated using Lipofectamine 3000 (Invi-

trogen). GFP expression was monitored. At 24 h post-

transfection, the cells were treated with 10 μM HU-1a or 10

μM 1E7-03 as indicated. At 48 h post-transfection, the cells

were lyzed in luciferase buﬀer using Lucilite kit (PerkinElmer).

n

followed by MS scans by collision-induced dissociation (CID)

S

https://dx.doi.org/10.1021/acsinfecdis.0c00511

ACS Infect. Dis. XXXX, XXX, XXX−XXX

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Article

activation mode. The precursor ions were fragmented with

collision energy of 18 with activation Q of 0.25 and an

activation time of 30 ms. Dynamic exclusion was enabled with

a repeat count of 2, a repeat duration of 15 s, exclusion

duration of 20 s, and an exclusion list size of 50.

Metabolic Stability in Plasma and Liver Microsomes.

HU-1a or 1E7-03 were dissolved in DMSO to obtain 10 mM

stock solution and mixed with human or mouse plasma

in 300 μL of a labeling solution consisting of solution A (PBS)

and solution B (0.2 M sodium bicarbonate solution pH 9.0)

with a ratio (20:1, A:B) at a ﬁnal concentration of 10 nM.

Group iii was mixed with 6 μL of 10 mM 1E7-03 in DMSO at

room temperature for 30 min, while groups i and ii were mixed

with the same volume of DMSO. Next, 20 μL of ELNLLB

(EZ-Link NHS-LC-LC-Biotin, 21343, Thermo Scientiﬁc) was

added into groups ii and iii and incubated for another 30 min,

and DMSO was added into group i as control. Subsequently,

(

H3667, M5905, Sigma-Aldrich) to a ﬁnal concentration of 10

μM and incubated at 37 °C. Aliquots were collected at

diﬀerent time points for up to 6 h. The resulting sample (50

μL) was mixed with 200 μL of cold acetone, vortexed for 2

min, and kept at −20 °C for 30 min, and then the precipitated

protein was removed by centrifugation at 13 000g for 5 min.

The supernatant was transferred to a clean test tube and

evaporated to dryness using a SpeedVac concentrator. The

residue was reconstituted in 50 μL of acetonitrile for LC/FT-

MS analysis. The metabolic stability of HU-1a and 1E7-03 was

also investigated using a pool of human and mouse liver

microsomes (HMS9PL, MSS9PL, Thermo Scientiﬁc) follow-

ing the manufacturer’s instructions. In brief, incubations were

conducted at 37 ± 1 °C in mixtures containing 10 μL of

human or mouse liver microsomes (20 mg/mL), 366 μL of

potassium phosphate buﬀer (pH 7.4, 100 mM), and 4 μL of

1

E7-03 compound and painting molecule ELNLLB were

removed by centrifugation through ﬁlters (OD003C34, Life

Sciences) at 12 000 rpm for 2 min followed by ﬁve washes with

200 mM Ammonium Bicarbonate. The solutions on the top of

the ﬁlters were collected and adjusted to 200 μL. All samples

were denatured and reduced in 10 mM dithiothreitol for 1 h at

37 °C, alkylated with 30 mM iodoacetamide for 20 min at

room temperature in the dark, and digested for 3 h with 2 μg

of trypsin (Promega) at 37 °C on a dry bath. Tryptic peptides

were puriﬁed by Zip-Tip (Millipore) following manufacturer’s

instructions and analyzed by nano LC-FT/MS. Label-free

quantiﬁcation of peptides derived from PP1 was performed

with SIEVE 2.1 software (Thermo Scientiﬁc). The identiﬁed

peptides of PP1 by Proteome Discoverer 1.4 (Thermo

Scientiﬁc) were unloaded as framing seeds.

1

E7-03 or HU-1a (10 mM sock solution of 1E7-03 or HU-1a

Molecular Docking. The web server https://bio-hpc.

ucam.edu/achilles/ was used for docking simulation of 1E7-03

binding to PP1. The crystal structure of PP1 (PDB code:

in DMSO was diluted into 1 mM by PBS). The mixture was

pre-incubated for 5 min, the reactions were then initiated with

the addition of 20 μL of 20 mM NADPH (Sigma-Aldrich).

Aliquots (50 μL for each sample) were collected at diﬀerent

time points for up to 24 h. The resulting samples were mixed

with 200 μL of cold acetone, vortexed for 2 min, and kept at

3

E7A) was retrieved from the PDB database. The receptor was

prepared by the standard protocol in Discovery Studio v2.5

Accelrys Software Inc., United States). The water molecules

(

were removed, and hydrogen atoms were added to the whole

protein structure. The tool performs a series of docking

calculations across the protein surface in order to ﬁnd the spots

with best binding aﬃnities. After the aﬃnities are calculated,

the tool clusters the results according to spatial overlapping of

the resulting positions. For each cluster, the position with the

best aﬃnity is taken as the representation. The molecular

docking of HU-1a within the RVxF-binding site of PP1 was

carried out on GOLD 3.0.1 using the default settings of 10 GA

runs. The active site was deﬁned as all residues within 11 Å of

the cognate ligand 1E7-03. The non-bound parameters were

taken from the default cutoﬀ values, i.e., 2.5 Å for hydrogen

bonds and 4.0 Å for van der Waals interactions. When the top

three solutions attained RMSD values of less than 1.5 Å, the

docking calculation was terminated. All ﬁgures were prepared

using PyMOL 1.5.

−

20 °C for 30 min, and then the precipitated protein was

removed by centrifugation at 13 000g for 10 min. The

supernatant was transferred to a clean test tube and evaporated

to dryness using a SpeedVac concentrator. The residue was

reconstituted in 50 μL of acetonitrile with 0.1% FA for LC/

FT-MS analysis. All experiments were run in parallel in

triplicate.

PP1α Expression and Puriﬁcation. Recombinant PP1α

2

0

was prepared as previously described.

BL21 (DE3)

Escherichia coli cells (Invitrogen, Carlsbad, CA) were co-

transformed with a vector RP1B-PP1α7-300, which expresses

human PP1α (residues 7−300), and pGR07 (Takara), which

expresses GroEL/GroES chaperones. The cells were grown in

media supplemented with 1 mM MnCl at 30 °C to an A

≈

2

600

0

.5. Then arabinose (2 g/L) was added to induce expression of

the GroEL/GroES chaperones. When A₆₀₀≈ 1 was reached,

the cells were transferred to 10 °C and PP1α expression was

induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside

for 20 h. Harvested cells were lysed using high-pressure

homogenizer Avestin Emulsiﬂex C3 in a solution containing 50

mM Tris-HCl (pH 8.0), 5 mM imidazole, 700 mM NaCl, 1

NanoBiT Assay. Plasmids including the human PP1, PP1

mut (Y70W, L73Y, G274E, and A299E), cdNIPP1 (Residues

140-225), cdNIPP1 RATA (V201A and F203A), and cdNIPP1

helix mut (D164A, T171A, N174A, K175A, and I177A)

coding sequences were obtained from Addgene. Genes of

interest were subcloned into pFC34K vector to generate the C-

terminal LgBiT-fused PP1/PP1 mut and C-terminal SmBiT-

fused cdNIPP1/cdNIPP1 RATA/cdNIPP1 helix mut using

Sgﬂ plus Pmel restriction sites. HEK293T cells were plated in a

96-well white/clear culture plates with 40% conﬂuence and

transiently transfected with the indicated constructs (1:1 ratio

of interacting pairs) using Lipofectamine 3000 Plus in OPTI-

MEM as directed by the manufacturer. At 24 h post

transfection, cells were treated with serial concentrations

(1.3−14 μM) of HU-1a for an additional 6 h. Nano-Glo Live

Cell Substrate (N2012, Promega) was added, and lumines-

mM MnCl , 0.1% Triton X-100 (v/v), and protease inhibitors.

His-tagged PP1 was puriﬁed using a Ni-NTA IMAC column

2

(

Qiagen, Valencia, CA). PP1 was further puriﬁed using size

exclusion chromatography in SEC buﬀer (20 mM Tris pH 8.0,

00 mM NaCl, 0.5 mM TCEP, 1 mM MnCl ). Glycerol (50%

5

2

v/v) was added to the puriﬁed PP1, and the PP1 was

aliquoted, ﬂash frozen, and stored at −80 °C.

Protein Painting Analysis of 1E7-03 Binding to PP1.

20

We followed our recently described procedure, in which

three groups, i, ii, and iii, of molecular painting reactions were

set up. In each group, recombinant protein PP1 was dissolved

T

https://dx.doi.org/10.1021/acsinfecdis.0c00511

ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases

pubs.acs.org/journal/aidcbc

Article

cence was measured using a GloMax-Multi Detection System

ACKNOWLEDGMENTS

■

(

Promega).

We acknowledge Dr. Mathieu Bollen and Dr. Monique

Beullens for the gift of PP1 and GroEL/GroES chaperon

bacterial expression vectors. This work was supported by the

research grants from NIH to S.N. (P50HL118006,

R01HL125005, 5U54MD007597, AI117970, and

U19AI109664), to W.P. (1R01GM134683), and to X.G.

1R25DE025778). This research was also supported by a pilot

Statistical Analysis. All graphs were prepared using

GraphPad Prism 6 software. Data are presented as mean ±

SD or standard error of the mean (SEM) as indicated in the

ﬁgure legends. Means were compared with Student t-tests.

1

ASSOCIATED CONTENT

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acsinfecdis.0c00511.

■

(

*

award from the District of Columbia Center for AIDS

Research, an NIH funded program (AI117970), to X.L.

PP1-derived peptides that were not or weakly protected

by 1E7-03 from ELNLLB painting (Figure S1); purity

of compounds HU-1a, HU-1c, and HU-1d analyzed by

LC/MS analysis (Figure S2) (PDF )

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Structural Optimization of 2,3-Dihydro-1H-cyclopenta[ b]quinolines Targeting the Noncatalytic RVxF Site of Protein Phosphatase 1 for HIV-1 Inhibition

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