Synthesis, Antiviral Activity, and Structure–Activity Relationship of 1,3-Benzodioxolyl Pyrrole-Based Entry Inhibitors Targeting the Phe43 Cavity in HIV-1 gp120

DOI: 10.1002/cmdc.201800534

Source and publish data:

ChemMedChem p. 2332 - 2348 (2018)

Update date:2022-08-16

Topics:

Authors:

Curreli, Francesca

Belov, Dmitry S.

Ahmed, Shahad

Ramesh, Ranjith R.

Kurkin, Alexander V.

Altieri, Andrea

Debnath, Asim K.

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Article abstract of DOI:10.1002/cmdc.201800534

The pathway by which HIV-1 enters host cells is a prime target for novel drug discovery because of its critical role in the life cycle of HIV-1. The HIV-1 envelope glycoprotein gp120 plays an important role in initiating virus entry by targeting the primary cell receptor CD4. We explored the substitution of bulky molecular groups in region I in the NBD class of entry inhibitors. Previous attempts at bulky substituents in that region abolished antiviral activity, even though the binding site is hydrophobic. We synthesized a series of entry inhibitors containing the 1,3-benzodioxolyl moiety or its bioisostere, 2,1,3-benzothiadiazole. The introduction of the bulkier groups was well tolerated, and despite only minor improvements in antiviral activity, the selectivity index of these compounds improved significantly.

Full text of DOI:10.1002/cmdc.201800534

Accepted Article

Title: Synthesis, antiviral activity, and structure-activity relationship

of 1,3-benzodioxolyl pyrrole-based entry inhibitors targeting the

Phenyl43 cavity in HIV-1 gp120

Authors: Asim K Debnath, Francesca Curreli, Dmitry S Belov, Shahad

Ahmed, Ranjith R. Ramesh, Alexander V Kurkin, and Andrea

Altieri

This manuscript has been accepted after peer review and appears as an

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to this Accepted Article as a result of editing. Readers should obtain

the VoR from the journal website shown below when it is published

to ensure accuracy of information. The authors are responsible for the

content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.201800534

Link to VoR: http://dx.doi.org/10.1002/cmdc.201800534

A Journal of

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ChemMedChem

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ChemMedChem

Synthesis, antiviral activity, and structure-activity relationship of 1,3-benzodioxolyl

pyrrole-based entry inhibitors targeting the Phenyl43 cavity in HIV-1 gp120

--Manuscript Draft--

Manuscript Number:

Article Type:

cmdc.201800534R1

Full Paper

Corresponding Author:

Asim K Debnath, Ph.D.

New York Blood Center

New York, NY UNITED STATES

Corresponding Author E-Mail:

adebnath@nybc.org

Order of Authors (with Contributor Roles): Asim K Debnath, Ph.D.

Francesca Curreli

Dmitry S Belov

Shahad Ahmed

Ranjith R. Ramesh

Alexander V Kurkin

Andrea Altieri

Keywords:

Abstract:

HIV-1. ENV-pseudovirus. virus entry antagonist. structure-activity relationship (SAR).

Reverse transcriptase (RT)

The pathway by which HIV-1 enters host cells is a prime target for novel drug discovery

because of its critical role in the HIV-1 life cycle. The HIV-1 envelop glycoprotein gp120

plays an important role in initiating virus entry by targeting the primary cell receptor

CD4. We explored the substitution of bulky molecular groups in region I in the NBD

class of entry inhibitors. Previous attempts at bulky substitutions in that region

abolished the antiviral activity, even though the binding site is hydrophobic. We

synthesized a series of entry inhibitors containing 1,3-benzodioxolyl moiety or its

bioisostere, 2,1,3-benzothiadiazole. The introduction of the bulkier groups was well

tolerated, and despite only minor improvements in antiviral activity, the selectivity index

(SI) improved significantly.

Response to Reviewers:

Response to the comments by the reviewers

Manuscript number: cmdc.201800534

MS Type: Full Paper

Title: "Synthesis, antiviral activity and Structure-activity relationship (SAR) of the 1,3-

benzodioxolyl pyrrole-based entry inhibitors targeted to the Phenyl43 cavity in HIV-1

gp120"

Correspondence Author: Dr. Asim K Debnath

COMMENTS TO AUTHOR:

Reviewer 1:

.The authors should fix "…we tried to introduce -C(=O)CH3 at the…" as superscript of

in page 1.

Response: It was corrected.

2.The authors should fix "…compared to NBD-556 and YYA-02 (Figure 1)…" into

"…compared to NBD-556 and YYA-021 (Figure 1)…" in page 2.

Response: The error was corrected.

ChemMedChem

10.1002/cmdc.201800534

3.The authors should refer to the following paper concerning YYA-021.

Y. Yamada, C. Ochiai, K. Yoshimura, T. Tanaka, N. Ohashi, T. Narumi, W. Nomura, S.

Harada, S. Matsushita, H. Tamamura, Bioorg. Med. Chem. Lett. 2010, 20, 354-358.

Response: The reference has been changed as suggested.

3.The authors should exchange from "…was observed by Mizuguchi et al. [6a]. We

further…" to "…was observed by Ohashi, et al. [6a]. We further…" in page 5, because

the first author should be picked up.

Response: The sentence was corrected as per the suggestion.

4.The authors should describe ratio of diastereoselectivity in obtained compounds 32

and 35 utilizing NMR or HPLC. If the ratio of diastereoselectivity cannot be detected,

the authors should comment the reason why.

Response: Compounds 32 and 35 were obtained as inseparable mixtures of

diastereomers with a diastereomeric ratio (dr) of about 4:1 and 3:1 (dr calculated by the

NMR different signal ration at the C1, since they differ in configuration at C1).

5.In Table 2, activity of compounds is almost the same against pseudoviruses of

several subtypes. Is it OK? Can the authors comment the reason why?

Response: We have added the possible reason for the similar antiviral activity of

compounds in Table 2.

Reviewer 2: Comments:

1)Scheme 3: Target structure does not have R2 group.

Response: We recognize that the target structures do not have R2 but the

intermediate 37 has a varied R2 groups.

2)Some structures (55 and 56) are not visible in the Table

Response: The column area of Table 1 has been expanded so that all structures are

now visible.

3)Author should mention the design rationale to add two methyl groups adjacent to the

primary amine group.

Response: We have now added the reason of adding two methyl groups at the α-

carbon atom of the primary amine.

4)In the structure table, R and S assignment cannot be done unless it was determined.

Authors should instead refer them as enantiomer 1 and enantiomer 2.

Response: We would like to thank the reviewer for the suggestion. Now Table 1

reflects this change.

5)The compounds presented in this manuscript seem to be dual (entry inhibitors and

RT inhibitors) targeting compounds. Therefore, a detailed discussion is required.

Response: We also previously reported that some NBD series compounds have

moderate antiviral activity against the HIV-1 RT. We also showed the same effect in

this series. We have discussed that in the results & Discussions section.

6)Since the design strategy mention about exploitation of Phe pocket, it is reasonable

to show their binding to target protein.

Response: We have presented docking based results in Figure 2.

ChemMedChem

10.1002/cmdc.201800534

Reviewer 3:

Major changes:

1.There appears to be inconsistencies regarding the determination of toxicity. Toxicity

should be shown for 14113, 14110, 14123, and 14159 in the Cf2Th-CCR5 cells and

the U87-CD4-CCR5 cells, 2. From the toxicities that are reported, there is a differential

between cell types. This is to be expected as cell lines are cancer cells and have

different mutations that cause their cancer phenotype. To make the reduction of toxicity

of the reported compounds, which is a major point of the study, more physiologically

relevant, the toxicities should be performed in a primary human target cell, either

PBMCs, macrophages or T-cells. To go along with this, the main compounds

mentioned should be tested against at least one isolate infecting the chosen cell type.

Response: All toxicity data are now provided as suggested.

2.The assumption throughout the manuscript is that the changes to the molecules have

not changed the target region of the compounds, namely the Phe43 pocket. Although

the difference in VSVG vs HIV-1 Env potencies allude to Env being the target, it would

drastically improve the study if this was demonstrated. This reviewer suggest to

maintain the virus based assay bias in the study and use a virus pseudotyped with an

Env containing the S375W mutation - if the compounds still target the Phe43 cavity,

this mutation should block the binding and reduce the compounds potency

significantly. Again, this need only be done with the top compounds, and would allow

the authors to include some modelling. To expedite matters, this experiment could be

combined with the experiment asked for above.

Response: Recently, we have shown (Europ. J med Chem, 154, 367, 2018) that the

HIV-1 mutants S375Y and S375W did not show any resistance to the NBD series

compounds with a phenyl ring in Region I. In this study, we have now tested this series

of NBD compounds against these two mutants and got similar results.

Minor changes (but important):

1.The intro needs to be rewritten to make it smoother, more concise, and highlight the

overall goal and rationale in a more clear manner. Similarly, the rest of the manuscript

would benefit from improvements in writing style.

Response: A Science Editor edited the entire revised manuscript except for the

Experimental part. This has substantially improved the writing style.

2.Just a suggestion but since the authors have two metrics for specificity of the

compounds, CC50/IC50 and VSVG IC50/HIV-1 IC50, term selectivity index is

confusing and it is not clear which of these is being referred to. Therefore, suggestions

selectivity index refers to specificity to HIV-1 Env, and the toxicity metric is referred to

as the therapeutic index.

Response: With due respect to the reviewer’s comments, we could not take this

suggestion because the therapeutic index is referred to as the ratio the dose that

causes toxic effect and the dosage that causes a therapeutic effect. Referring Toxicity

metric as the therapeutic index may be misleading.

Section/Category:

Additional Information:

Question

Response

Yes

Submitted solely to this journal?

Has there been a previous version?

Do you or any of your co-authors have a No. The authors declare no conflict of interest.

conflict of interest to declare?

ChemMedChem

10.1002/cmdc.201800534

Animal/tissue experiments?

Additional Material - Author

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10.1002/cmdc.201800534

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Additional Material - Author

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FULL PAPER

Synthesis, antiviral activity, and structure-activity relationship of

,3-benzodioxolyl pyrrole-based entry inhibitors targeting the

Phenyl43 cavity in HIV-1 gp120

Francesca Curreli , Dmitry S Belov , Shahad Ahmed , Ranjith R. Ramesh , Alexander V Kurkin ,

Andrea Altieri and Asim K Debnath

[

^aLaboratory of Molecular Modeling & Drug Design, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E 67^t^hStreet, New York, NY 10065,

USA

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

Supporting information for this article is given via a link at the end of the document.

Abstract: The pathway by which HIV-1 enters host cells is a prime

early attempts were promising, there has since been only limited

exploration of substituent variations in the phenyl ring of NBD-

target for novel drug discovery because of its critical role in the HIV-

556 and its analogs.

life cycle. The HIV-1 envelop glycoprotein gp120 plays an

important role in initiating virus entry by targeting the primary cell

receptor CD4. We explored the substitution of bulky molecular

groups in region I in the NBD class of entry inhibitors. Previous

attempts at bulky substitutions in that region abolished the antiviral

activity, even though the binding site is hydrophobic. We synthesized

a series of entry inhibitors containing 1,3-benzodioxolyl moiety or its

bioisostere, 2,1,3-benzothiadiazole. The introduction of the bulkier

groups was well tolerated, and despite only minor improvements in

antiviral activity, the selectivity index (SI) improved significantly.

We recently explored methoxy substitution in both the meta- and

the para- positions of the phenyl ring of a new lead compound,

NBD-14010, but both substitutions completely abolished the

[4]

antiviral activity . We hypothesize that the bulky and flexible

nature of the methoxy substituent might be responsible for the

loss of activity. To overcome the effect of the flexible substituent

in region I, we decided to use 1,3-benzodioxole and its

[5]

bioisostere 2,1,3-benzothiadiazole , which are more compact

but bulkier than the methoxy substituent. Mizuguchi et al. in

2016 used

a 1,3-benzodioxole moiety to replace a 4-

chlorophenyl group of

Introduction

The development of anti-HIV-1 drugs with known and new

targets, the reduction of the side effects of antiviral therapies,

and, most critically, the optimization of antiretroviral combination

therapies are helping many patients with HIV-1 infection to live a

healthy life. Despite those successes, anti-HIV-1 drugs are still

hampered by toxicities and drug resistance. Patient compliance

with drug regimens also remains an issue. More research is

needed to develop drugs with novel targets that block the HIV-1

replication cycle and work by different mechanisms than the

currently available drugs.

Despite concerted efforts over the last two decades to develop a

drug that blocks HIV-1 entry into host cells by preventing the

HIV-1 envelop glycoprotein gp120 from binding to the cellular

receptor CD4, there is no such drug yet available on the market.

In 2015, the drug BMS-663068 (Fostemsavir) received the

“

Breakthrough Therapy” designation from the US FDA,

representing a potential success in the long search for a drug

that targets gp120. In 2017, ViiV Healthcare, the developer of

Fostemsavir, announced positive phase 3 results from the

BRIGHTE study of heavily treated patients with HIV infection.

[6]

Figure 1. The chemical structure of NBD-556, YYA-021 , and our

[1a, 4, 7]

next-generation lead compounds

Since 2005, our group has sought to develop HIV-1 host cell-

entry inhibitors by targeting the Phe43 cavity in gp120. We have

designed many potent gp120 antagonists since the discovery of

NBD-556 (Figure 1), which acts as a CD4 mimic and gp120

agonist . Because of the narrow space in the Phe43 cavity, the

phenyl ring of NBD-556-type CD4 mimics will not tolerate a

bulky substituent. For example, when we tried to introduce –

NBD-556. The resulting compound had about 10-fold less

antiviral activity compared with NBD-556 and was also more

toxic .

[

We have developed a set of new lead compounds (NBD-11021,

NBD-14010 and NBD-14189) with markedly different structural

C(=O)CH at the para- position of the phenyl ring, the antiviral

profiles than NBD-556 and YYA-021^[

(Figure 1). We

activity was lost. Similarly, a bulkier group such as cycloheptyl in

[

hypothesize that the structures of the new lead compounds

might allow them to tolerate the introduction of 1,3-benzodioxole

place of phenyl also completely abolished the antiviral activity .

We also attempted to introduce fluorine at the meta- position of

[5]

_t_h_e_p_h_e_n_y_l_r_i_n_g_,_b_u_t_w_i_t_h_o_u_t_s_u_c_c_e_s_[2-3]. Because none of the

or its bioisostere 2,1,3-benzothiadiazole , resulting in retained

FULL PAPER

ChemMedChem

10.1002/cmdc.201800534

[

antiviral activity, improved toxicity profile, and higher selectivity

index (SI).

haloform reaction . Aryl bromides 4 and 8 were commercially

available. Aryl bromides 13 and 22 were prepared using

procedures from the literature ^[¹⁰^].

Protected amines 37, 39, 40, 41, and 42 and protected

thiazoles 31 and 34 were prepared as described previously^[

⁷^c^,¹¹^]. Two protected amines, 33 and 36, with the gem-dimethyl

moiety were prepared using 1,2-addition of thiazolyl anions of

the two enantiopure imines 30, as per Scheme 2. Compounds

Here, we synthesized 22 entry inhibitors with substitutions

4, 7b,

of 1,3-benzodioxole or its bioisostere to generate

comprehensive understanding of the structure-activity

relationship (SAR) of these bulky substituents in region I of the

phenyl ring of NBD-type CD4 mimics.

2 and 35 were obtained as inseparable mixtures of

diastereomers with dr~4:1 - 3:1 (differs in configuration at C1,

based on NMR). Because of the presence of acidic N-H amide,

we used 2.5 equivalent of metallated thiazole. The absolute

configuration of compounds 33 and 36 was not determined, and,

as in prior works, compounds derived from (enantiomer 2)-30

were designated as A, and compounds derived from

enantiomer 1)-30 were designated as B. We prepared the final

library of NBD compounds using peptide coupling, followed by

_p_r_o_t_e_c_t_i_n_g_-_g_r_o_u_p_c_l_e_a_v_a_g_e₍_S_c_h_e_m_e₃₎[12]

Results and Discussion

Chemistry

The synthesis of the novel NBD compounds is described in

Schemes 1-3. First, we prepared a series of carboxylic acids

with 1,3-benzodioxole or its bioisostere 2,1,3-benzothiadiazole

(

Scheme 1). Aniline 1 was acylated with ethyl 2-chloro-2-

[2]

oxoacetate and after saponification yielded acid 3 . Acids 18-

0, 26 were prepared using a general scheme involving Suzuki

coupling of aryl bromides 4, 8, 13, and 22 with N-Boc-2-pyrrole

boronic acid, followed by Boc cleavage, acylation, and semi-

Scheme 1. Synthesis of 1,3-benzodioxole and its bioisostere containing acids. *yields are over two steps.

FULL PAPER

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10.1002/cmdc.201800534

Scheme 2 Synthesis of protected thiazole ring-containing amines.

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ChemMedChem

10.1002/cmdc.201800534

Scheme 3. Synthesis of final NBD compounds.

[

Antiviral screening and SAR analysis

previously . As a control, we used NBD-14113, which contains

the sterically smallest substituent fluorine (F) at the meta- and

para- positions. NBD-14110, which has the 1,3-benzodioxole

moiety in region I, showed a slight improvement in antiviral

activity in both the single-cycle and the multi-cycle assays and

also a ~1.7-fold improvement in cytotoxicity (CC50) compared

with the control. NBD-14111 lost its antiviral activity, although it

still had reduced cytotoxicity compared with the control. We

We evaluated the HIV-1-inhibitory activity and cytotoxicity of the

NBD compounds. We assessed the anti-HIV-1 activity in a

single-cycle assay by infecting TZM-bl indicator cells with the

pseudovirus HIV-1HXB2. We also assessed the anti-HIV-1

activity in a multi-cycle assay by infecting MT-2 human T-cells

with the full-length, lab-adapted virus HIV-1IIIB, as described

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ChemMedChem

10.1002/cmdc.201800534

substituted the hydrogens of the α-carbon of the ethylamine

group in NBD-14110 and NBD-14111 with two methyl groups to

understand the SAR effect of the bulk in that region. The

resulting compounds (NBD-14116 and NBD-14117) showed

similar antiviral activity but became more cytotoxic. When we

removed the methyl group from the thiazole of NBD-14110 to

convert it to NBD-14122, we observed a ~1.9-fold reduction in

antiviral activity. However, the antiviral activity of NBD-14123

the antiviral activity was completely lost, unlike what was

observed by Ohashi et al. ^[⁷^a^]. We attempted to replace the 1,3-

benzodioxole

moiety

with

its

bioisostere,

2,1,3-

[

benzothiadiazole . The resulting NBD-14127 showed ~1.6-fold

improvement in antiviral potency compared with NBD-14123,

and NBD-14126 showed about 2.8-fold improvement in antiviral

activity compared with NBD-14122. The cytotoxicity of both

NBD-14126 and NBD-14127 was higher by about 1.5-fold

compared with that of the parent compounds.

(

converted from NBD-14111) remained similar to that of NBD-

4111, and there was no noticeable change in cytotoxicity. The

The SAR analysis indicated that bulkier groups such as

1,3-benzodioxole or its bioisostere 2,1,3-benzothiadiazole^[⁵^]are

well tolerated to replace the phenyl moiety in region I. This was

also validated by docking one of the best leads, NBD-14110, in

the Phe43 cavity of HIV-1 gp120 (Figure 2) using Glide software

from Schrodinger (Cambridge, USA). When we examined the SI

addition of a methyl group in the primary amine of NBD-14139

and NBD-14140 resulted in marginal improvement of antiviral

[

SI = CC50/IC50)], NBD-14110, NBD-14123, and NBD-14159

turned out to be the best leads for further exploration.

Antiviral activity of the polycyclic NBD compounds against

a large panel of HIV-1 Env-pseudotyped reference viruses.

We selected the three new NBD compounds (NBD-14110, NBD-

14123, and NBD-14159) based on their anti-HIV-1 activity and

the SI after comparing data from the single-cycle and multi-cycle

cell-based assays. We evaluated those compounds against a

selection of 34 HIV-1 clones of clinical isolates of subtypes A, B,

C, and D and of recombinant subtype A (Arec), including A1/D,

A2/D, and AG. We compared the three selected compounds

with the previously described compound NBD-14113^[. We

found that the three polycyclic compounds exhibited anti-HIV-1

activity at low micromolar concentrations, with overall mean IC50

values very similar to that of NBD-14113 (Table 2). When we

analyzed the data obtained from the different viral subtypes, the

mean IC50 for NBD-14113 across the subtype Arec and subtype

C viruses was slightly higher than that across the subtype A, B,

and D viruses. NBD-14110 was equally active against all the

viral subtypes, as indicated by the similar mean IC50 values

obtained for that compound. NBD-14123 and NBD-14159 were

slightly more active against the subtype A, Arec, B and D viruses

than against subtype C viruses, as shown by the mean IC50

values. Moreover, NBD-14110, NBD-14123, and NBD-14159

were weak inhibitors of the control pseudovirus VSV-G,

indicating that the inhibitory activity of these compounds is

specific to HIV-1. We observed similar activity of these three

molecules against all the clinical isolated tested. We reason that

despite possible sequence difference in the Phe43 cavity of the

isolates tested, the variation in substituents in the molecule are

loated in the α-carbon atom of the primary amine and the

13]

Figure 2. 2D-interaction of NBD-14110 with HIV-1 gp120 in the

Phe43 cavity based on Glide-based docking.

activity; however, the cytotoxicity remained the same.

Interestingly, when we replaced the hydrogens of the α-carbon

of the ethylamine of NBD-14123 with two methyl groups, the

antiviral activity of NBD-14119 was improved by ~2.3-fold, while

that of NBD-14118 remained similar to that of NBD-14116; the

cytotoxicity of both NBD-14118 and NBD-14119 was higher, as

was observed with NBD-14117 and NBD-14118.

We further explored the SAR by adding di-fluoro at position

of the 1,3-benzodioxole moiety. NBD-14144 showed ~4.5-fold

higher antiviral activity compared with NBD-14122. NBD-14145

showed less improvement (~1.7-fold) in antiviral activity. It is

worth noting, however, that the cytotoxicity of both compounds

became much higher (~3.9-fold). When we added two methyl

groups at position 2 of the moiety, NBD-14133 and NBD-14134

thiazole ring (CH vs. H) which have least impact in the binding

of the molecules in the cavity as demonstrated in Figure 2.

Additionally, we tested the anti-HIV-1 activity of the new

polycyclic NBD compounds against a set of paired infant and

maternal HIV-1 Env molecular clones of subtype A and C/D,

which were isolated from infants infected between birth and 6

weeks postpartum and from the respective chronically infected

lost their antiviral potency. The positional switching of CH OH in

NBD-14172 improved the antiviral potency by about 1.5-fold

compared with that of NBD-14123, whereas similar switching in

NBD-14122 reduced the antiviral activity of the resultant

[

14]

mothers

. The infant variants were poorly neutralized by

combinations of Mab 2G12, biz, 2F5, and 4E10 ^[. We detected

no differences in the activity of the NBD compounds against the

vertically transmitted viral types, indicating that all the polycyclic

compounds neutralized the infant and maternal HIV-1 variants

equally (Table 3). NBD-14159 was the most effective compound

14]

(

enantiomer 1)enantiomer NBD-14173 by ~1.9 fold; the

cytotoxicity was higher for both compounds, however. Addition

of a methyl group in the primary amine of NBD-14158 and NBD-

14159 did not have any effect on antiviral potency or cytotoxicity.

When we attempted to introduce the 1,3-benzodioxole

moiety in the oxalamide-containing NBD-14108 and NBD-14109,

FULL PAPER

ChemMedChem

10.1002/cmdc.201800534

with an overall mean IC50 of 3.9±0.3 µM. NBD-14110 was the

least effective with an overall mean IC50 of 5±0.5 µM.

Taken together, these results suggest that the HIV-1 entry

antagonist property is maintained in these new generation NBD

compounds as well (Figure 3).

Antiviral activity of the polycyclic NBD compounds against

three HIV-1 isolates in human PBMC.

Because some NBD entry antagonists have shown

[

The new polycyclic NBD compounds were tested in human

PBMC cells against 3 competent HIV-1 isolates. We used two

lab-adapted viruses, the CXCR4-tropic HIV-1LAI, and the CCR5-

tropic HIV-1BaL and the CCR5-tropic HIV-197USSN54, primary

clinical isolate of subtype A. We found that all the NBD

compounds neutralized the lab-adapted viruses with similar

antiviral potency as shown by the IC50 in range of 1.9-3.8 µM

moderate activity against HIV-1 reverse transcriptase (RT) , we

evaluated the activity of the polycyclic NBD compounds against

that enzyme. We used NBD-556 and Nevirapine as controls. As

previously reported, 300 μM NBD-556 had no activity against

HIV-1 RT^[¹³^,¹⁵^], whereas Nevirapine was extremely potent

against HIV-1 RT, with an IC50 of 0.20 μM (Table 4). All of the

polycyclic NBD compounds inhibited HIV-1 RT with an IC50 in

the range of 28–45 μM. Those IC50 values are more comparable

to the values obtained by testing the NBD compounds against

the control pseudovirus VSV-G than to those obtained in the

HIV-1 neutralization assays, suggesting that the HIV-1 RT is not

the primary target of the polycyclic NBD compounds.

(

Table 4). NBD-14110 showed a slightly better activity against

the primary isolate HIV-197USSN54. The cytotoxicity assay

indicated that the CC50 for these compounds tested in the

human PBMC was ≥ 47 µM.

NBD compounds do not enhance HIV-1 entry into CD4-

negative cells and showed moderate activity against HIV-1

reverse transcriptase (RT)

S375Y and S375W mutants are not resistant to NBD

compounds

We evaluated whether the selected polycyclic compounds NBD-

Previously, we reported that some specific amino acid

substitutions in the CD4-binding site of the ENV gp120 rendered

the mutant virus resistant to the NBD compounds, but S375H,

S375W, and S375Y mutant viruses were not. In this study, we

tested NBD-14110 against the mutant pseudovirus HIV-1HXB2

carrying amino acid substitution S375Y and S375W in the ENV

gp120 region. This compound showed similar potency against

this mutant as was observed against HIV-1HXB2-WT (data not

shown). The data indicate that NBD-14110 is sensitive to the

4110, NBD-14123, and NBD-14159 support HIV-1 entry into

CD4-negative cells. We used NBD-556 (an HIV-1 entry agonist

^[¹³^,¹⁵^])

as a control. Although NBD-556 enhanced the infection of

the Cf2Th-CCR5 cells, we found that none of the new polycyclic

NBD compounds enhanced HIV-1 infectivity in those cells.

To exclude false negative results we performed a cytotoxicity

assay by treating these cells with escalating concentrations of

the above NBD compounds and we found that the CC50

detected for NBD-14113 was ~90 µM while NBD-556, NBD-

S375Y

and

S375W

mutant

viruses.

14110, NBD-14123 and NBD-14159 had a CC50 >100 µM.

1 0

N B D -5 5 6

N B D -1 4 1 1 3

N B D -1 4 1 1 0

N B D -1 4 1 2 3

N B D -1 4 1 5 9

C o n c e n tra tio n ( M )

Figure 3. Infectivity of CD4 negative Cf2Th−CCR5 cells by HIV-1ADA (CD4-dependent virus). Cf2Th−CCR5 cells were infected with HIV-1ADA in the presence of

the NBD entry inhibitors. The relative virus infectivity indicates the ratio between the amount of infection detected in the presence of the compounds and the

amount of infection detected in the absence of the compounds. Three independent experiments were performed in triplicate, and the graph is representative of

one experiment; the values represent the mean ± standard deviation.

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Table 1. Anti-HIV-1 activity (IC50) and cytotoxicity (CC50) of NBD compounds in single-cycle (TZM-bl cells) and multi-cycle (MT-2

cells) assays

TZMb-l

MT-2

Code (enantiomer)

Structure

IC (µM)

CC (µM)

IC (µM)

CC (µM)

NBD-14113

3.3±0.3a

2.3±0.1

7±0.4

84.1±1.3

145.6±7.6

145.8±5.2

55.2±5.4

66.9±1.4

154.4±1.5

142.3±2.6

25.5

4.8±0.8a

3.8±1.4

9.8±4.8

2.3±0.3

2.5±1

96.8±1.8

193±6.3

170±9

20.2

(

enantiomer 1)

NBD-14110

63.3

20.8

9.7

50.8

17.3

42.6

39.2

35.8

42.5

(

enantiomer 2)

NBD-14111

enantiomer 1)

NBD-14116

5.7±0.4

2.9±0.7

6.8±0.7

4.3±0.8

98±2.2

enantiomer 1)

NBD-14117 (enantiomer 2)

23.1

22.7

33.1

98±1.5

NBD-14122

5.1±0.4

182.8±3

(enantiomer 1)

NBD-14123

4.5±0.9

191.5±2.6

(enantiomer 2)

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NBD-14139

.6±0.6

149±11.3

153±11.5

32.4

41.3

7.5±1.7

178.1±8

173.2±8

23.7

(

enantiomer 2)

NBD-14140

3.7±1.3

9.32±2.8

18.5

(

enantiomer 1)

NBD-14118

5.4±0.2

64.3±3.7

11.9

4.3±2

123±10.5

28.6

(

enantiomer 1)

NBD-14119

1.9±0.4

69.2±3.8

36.4

2.8±0.9

106±4.4

37.8

(

enantiomer 2)

NBD-14144

1.5±0.1

37.9±2.4

25.2

2.7±0.2

43.4±3.6

16.1

(enantiomer 1)

NBD-14145 (enantiomer 2)

2.5±0.2

38.7±1.3

15.5

3.1±0.5

41±2.7

13.2

NBD-14133

64±1.2

16.2±0.4

15.3±5

96.5±1.2

89.3±1.3

5.9

5.8

(enantiomer 2)

NBD-14134

61.1±1.4

(enantiomer 1)

NBD-14172

2.8±1.3

92.3±2.9

32.9

2.4±0.3

156.1±14.3

65.0

(

enantiomer 2)

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NBD-14173

.4±2

105.2±7.9

12.5

8.2±2.7

176.1±4.3

21.5

(

enantiomer 1)

NBD-14158

8.1±1.1

157.3±7.8

19.4

9.5±0.5

192.4±4.2

20.2

(

enantiomer 1)

NBD-14159

2.7±0.3

95.8±1.4

35.5

2.98±0.1

180±2

60.4

(

enantiomer 2)

NBD-14108

>53

≥750

>79

≥650

(

enantiomer 2)

NBD-14109

≥750

>79

≥650

(

enantiomer 1)

NBD-14126

2.4±0.5

79.9±1

33.3

4±0.7

117.4±4.3

29.3

(

enantiomer 1)

NBD-14127

2.7±0.3

96.7±2.9

35.8

7.3±1.5

137±1.5

18.8

(

enantiomer 2)

Data previously reported ^[¹³^]; the reported IC50 and CC50 values represent the means ± standard deviation (SD), n=3.

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Table 2. Neutralization activity of NBD compounds against a panel of HIV-1 Env pseudoviruses

IC50 (µM)^a

Subtype

NIH #

ENV

NBD-14113

NBD-14110

NBD-14123

NBD-14159

11887

Q259ENV.W6

2.1±0.3

1.9±0.2

2.3±0.2

1.3±0.2

11888

QB726.70M.ENV.C4

QF495.23M.ENV.A3

BG505-T332N

±0.2

3.7±0.3

3±0.1

3.5±0.1

3.3±0.2

2.7±0.2

3.7±0.1

11891

±0.2

.3±0.2

.8±0.1

4.2±0.5

5.9±0.4

2.1±0.6

4±0.5

3.3±0.2

4±0.3

KNH1144

4.1±0.4

1.6±0.1

A1/D

11901

QA790.204I.ENV.A4

QA790.204I.ENV.C8

5.5±0.3

4.3±0.1

3±0.1

4±0.4

1903

1904

2.4±0.2

1.4±0.2

2.1±0.1

QA790.204I.ENV.E2

1.5±0.2

5.2±0.3

2.5±0.1

2.5±0.3

5.9±0.2

3.1±0.2

3.2±0.2

3.8±0.4

3.5±0.1

A2/D

11906

11595

11597

QG393.60M.ENV.B7

CRF02_AG, clone 251

CRF02_AG, clone 253

5.9±0.1

3±0.2

4.8±0.3

4.6±0.5

3.5±0.2

3.2±0.2

5.4±0.2

5±0.3

3.2±0.2

3.1±0.1

1599

1607

CRF02_AG, clone 257

CRF02_AG, clone 33

4.4±0.1

3.5±0.4

4.8±0.2

2.7±0.2

11018

1022

QH0692, clone 42

PVO, clone 4

.3±0.4

8.2±0.2

1.4±0.3

2.4±0.4

3.4±0.3

2.8±0.3

6.4±0.1

1.9±0.2

3.6±0.2

3.4±0.2

3.1±0.7

5.8±0.7

4.3±0.7

4.4±0.6

3.1±0.4

3.7±0.4

4±0.3

11023

11035

TRO, clone 11

.1±0.1

REJO4541, clone 67

.1±1

1036

1037

RHPA4259, clone 7

THRO4156, clone 18

1054.TC4.1499

.7±0.4

3.8±0.1

2.7±0.2

3.1±0.1

2.7±0.2

11561

.6±0.2

.2±0.2

.7±0.3

.4±0.1

±0.1

4.2±0.4

4.5±0.5

1.6±0.1

2±0.1

2.4±0.3

2.1±0.6

3.4±0.2

2.8±0.3

2.5±0.3

3±0.2

4.1±0.1

3.7±0.3

3.3±0.2

1.7±0.1

3.1±0.3

3.3±0.1

3.5±0.2

4.9±0.3

5.3±0.2

5.8±0.3

4.2±0.5

5±0.1

1572

9021_14.B2.4571

_W_E_A_U_d₁₅_.₄₁₀_.₅₀₁₇b

1578

B41

11308

Du422, clone 1

1.8±0.2

4.9±0.1

5.5±0.4

1.7±0.1

3.5±0.1

4.4±0.4

4.7±0.1

4.3±0.5

2.9±0.3

2.4±0.2

5.9±0.4

11314

11317

11500

ZM109F.PB4

.9±0.1

CAP210.2.00.E8

3.7±0.1

4.3±0.4

5.3±0.2

3.2±0.2

4.4±0.5

5.7±0.7

5.6±0.3

4.4±0.6

2.5±0.4

3.5±0.3

5.3±0.4

HIV-00836-2, clone 5

HIV-16936-2, clone 21

HIV-25711-2, clone 4

HIV-25925-2, clone 22

QB099.391M.ENV.C8

QA013.70I.ENV.H1

QD435.100M.ENV.B5

QD435.100M.ENV.E1

11504

11506

.9±0.2

.7±0.4

.8±0.3

±0.3

1507

1909

11911

.5±0.1

.2±0.3

.8±0.6

4.3±0.7

3.3±0.2

3.5±0.2

1916

1918

Control

VSV-G^c

26.3±1.4

3.9±0.17

51.6±4.4

45.3±2.3

41.3±3.4

Mean ± SEM (µM): Overall (n=34)

3.56±0.27

3.54±0.22

3.68±0.18

Subtype A (n=5)

Subtype Arec (n=8)

Subtype B (n=10)

Subtype C (n=7)

3.44±0.36

4.33±0.39

3.79±0.18

4.04±0.43

3.5±0.75

3.74±0.66

3.4±0.55

3.32±0.63

3.85±0.5

3.7±1.1

3.44±0.32

3.2±0.52

3.22±0.4

4.26±0.44

3.77±0.82

3±0.48

3.4±0.29

3.68±0.35

4.39±0.36

3.7±0.31

Subtype D (n=3)

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

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

VSV-G was tested in U87-CD4-CCR5 cells. The CC50 for NBD-14113 was ~75 µM, and the CC50 for NBD-14110,

NBD-14123 and NBD-14159 was >100 µM in these cells.

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Table 3. Neutralization activity of NBD compounds against an HIV-1 panel of “Paired Infant (B) and Maternal (M) Env

Molecular Clones.”

IC50 ₍_µ_M₎a

Subtype NIH #

ENV

NBD-14113 NBD-14110

NBD-14123

NBD-14159

11518-B

11528-M

BG505.W6M.ENV.C2

MG505.W0M.ENV.A2

4.4±0.3

4.7±0.8

4.8±0.3

6.1±0.5

4.4±0.1

3.7±0.2

4.7±0.5

4.6±0.1

11519-B

11531-M

B1206.W6P.ENV.A1A

MI206.W0M.ENV.D1

4.8±0.4

3.4±0.2

5.6±0.2

4.7±0.2

4.9±0.3

3.4±0.3

4±0.3

3.4±0.2

11521-B

11534-M

BJ613.W6M.ENV.E1

MJ613.W0M.ENV.A2

2.8±0.4

4.9±0.2

2.8±0.1

4.9±0.1

2.5±0.1

4±0.1

3±0.1

3.1±0.3

11525-B

11540-M

BL274.W6M.ENV.A3

ML274.W0M.ENV.B1

5.7±0.3

4.5±0.6

7.2±0.1

7.1±0.3

5.7±0.3

5.6±0.4

4.2±0.5

5.4±0.5

C/D

11522-B

11536-M

BK184.W6M.ENV.D2

MK184.W0M.ENV.E4

3.6±0.2

2.6±0.3

3.7±0.6

3.4±0.2

3.6±0.1

3.7±0.1

4.2±0.1

2.6±0.1

Mean ± SEM (µM): Overall (n=10)

4.1±0.3

4.3±0.5

4±0.4

5±0.5

4.2±0.3

4.2±0.6

4.1±0.4

3.9±0.3

4±0.3

Infant (B)

4.8±0.8

5.2±0.6

Mother (M)

3.8±0.5

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

Table 4. Anti-HIV-1 activity (IC50) and cytotoxicity (CC50) of

Table 5. Antiviral Activity of NBD Compounds

Polycyclic NBD compounds tested in human PBMC

against HIV-1 Reverse Transcriptase

Inhibitors

NBD-556

IC50 (μM)^a

>300

IC50 ₍_μ_M₎a

CC50

Inhibitors

HIV-

(μM)^a

HIV-1BaL

NBD-14110

NBD-14113

NBD-14123

30.6±2.7

45.9±3.6

39.5±2.8

LAI

97USSN54

NBD-14113

NBD-14110

NBD-14123

NBD-14159

3.8±1.1

3.5±0.9

2.9±0.9

2.6±0.7

3.8±0.4

2.8±0.6

1.9±0.2

2.8±0.4

3.1±0.5

1.1±0.3

1.8±0.6

3.6±0.4

47.1±2.6

62.8±2.9

61.3±4

NBD-14159

Nevirapine

28.1±7.9

0.2

68.3±5

The reported IC50 and CC50 values represent the

mean ± standard deviation (n = 3).

The reported IC50 values represent the means ± standard

deviations (n = 3).

Conclusions

We explored the effect of substituting a phenyl ring in region I of

the phenyl ring of NBD CD4 mimics with a bulkier moiety such

as 1,3-benzodioxole or its bioisostere. Three new NBD

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DCM (3x100 mL). The organic layer was dried over Na

evaporated. The residue was used without further purification.

2 4

SO and

compounds (NBD-14110, NBD-14123, and NBD-14159) with a

1,3-benzodioxole substituent exhibited the best improvement in

SI compared with the control, NBD-14113. Furthermore, those

inhibitors inhibited all of the tested clinical HIV-1 isolates with

similar potency. The 1,3-benzodioxole series compounds

showed poor activity against HIV-1 RT, indicating that they

primarily target gp120, thereby preventing HIV-1 entry into host

cells. We anticipate that these new lead compounds can be

further optimized to develop more potent and HIV-1 entry-

specific inhibitors.

2-(benzo[d][1,3]dioxol-5-yl)-1H-pyrrole (7)

Compound 7 was prepared according to the general procedure

for cross-coupling reaction and Boc cleavage from 4 (10.00 g).

M = 9.80 g. Yield >100% (over two steps).

¹H NMR (CDCl

H), 6.41 (br. s, 1 H), 6.81 - 6.85 (m, 2 H), 6.94 (dd, J=9.3, 1.2

Hz, 1 H), 6.98 (s, 1 H), 8.32 (br. s, 1 H).

, 400 MHz) δ = 5.98 (s, 2 H), 6.29 (q, J=2.7 Hz,

¹³C NMR (CDCl

110.1, 117.4, 118.5, 127.6, 132.3, 146.3, 148.3.

, 100 MHz) δ = 101.2, 105.3, 105.5, 108.8,

Experimental Section

2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-1H-pyrrole (10)

Compound 10 was prepared according to the general procedure

for cross-coupling reaction and Boc cleavage from 8 (8.43 g). M

= 8.15 g. Yield >100% (over two steps).

Synthesis

¹H NMR (CDCl

, 400 MHz) δ = 6.33 (q, J=2.7 Hz, 1 H), 6.46 -

.50 (m, 1 H), 6.85 - 6.89 (m, 1 H), 7.04 (d, J=8.9 Hz, 1 H), 7.12

7.17 (m, 2 H), 8.39 (br. s, 1 H).

ethyl 2-(benzo[d][1,3]dioxol-5-ylamino)-2-oxoacetate (2)

Aniline 1 (10.10 g, 73.6 mmol) was dissolved in CH

Et N (11.30 mL, 73.6 mmol) was added. To the resulted solution

ethyl chlorooxoacetate (9.05 mL, 73.6 mmol) was added

dropwise as a solution in CH Cl (74 mL) with cooling on a water

2 2

Cl (74 mL),

13_C_N_M_R₍_C_D_C_l

, 100 MHz) δ = 105.6, 106.5, 109.8, 110.3,

119.0, 119.5, 129.5, 131.0, 131.8 (t, J = 255.1 Hz), 142.2, 144.4.

bath. The reaction mixture was stirred for 1 hour and washed

with 5% HCl (~200 mL). The layers were separated, and the

2,2-dimethylbenzo[d][1,3]dioxole (12)

PBr (8.54 mL, 91 mmol, 0.4 equiv) was added dropwise to a

stirred solution of pyrocatechol (25 g, 227 mmol) and acetone

50 mL, 681 mmol, 3 equiv) in benzene (230 mL). The reaction

organic layer was extracted with CH

combined organic layers were dried over Na

(2 x 100 mL). The

SO , filtered and

(

evaporated. The residue was recrystallized from ethanol (50 mL)

to give 12.75 g (73%) of dirty-white solid.

mixture was stirred until HBr ceased to evolve (~4 hours) and

then poured into NaOH aqueous solution (100 g in 0.5 L). The

organic layer was separated, and the aqueous layer was

extracted with DCM (3x100 mL). The organic layer was dried

over Na SO and evaporated. The residue was dissolved in

2 4

hexane and filtered through a small plug of silica (~1 cm). The

¹H NMR (CDCl

, 400 MHz) δ = 1.29 (t, J=7.0 Hz, 3 H), 4.28 (q,

J=7.0 Hz, 2 H), 5.99 (s, 2 H), 6.87 (d, J=8.4 Hz, 1 H), 7.22 (d,

J=7.5 Hz, 1 H), 7.36 (s, 1 H), 10.66 (s, 1 H).

filtrate was evaporated to give a pure 12 as a colorless liquid. M

¹³C NMR (CDCl

113.7, 131.7, 144.1, 147.1, 155.1, 160.7.

, 100 MHz) δ = 13.8, 62.4, 101.2, 102.3, 108.0,

24.86 g. Yield = 73%.

1_H_N_M_R₍_C_D_C_l

, 400 MHz) δ = 1.70 (s, 6 H), 6.73 - 6.84 (m, 4

-(benzo[d][1,3]dioxol-5-ylamino)-2-oxoacetic acid (3)

The ester 2 (12.75 g, 53.80 mmol) was suspended in ethanol

108 mL), and a solution of NaOH (4.30 g, 108 mmol, 2.0 equiv)

H).

(

13_C_N_M_R₍_C_D_C_l

, 100 MHz) δ = 26.0 (2C), 108.6 (2C), 117.5,

in water (108 mL) was added. The resulting mixture was heated

at reflux for 2 days, cooled to room temperature and acidified

with conc. HCl (~10 mL). To the resulted homogeneous solution,

water was added until the product precipitates (~200 mL), and

the acid was filtered off. M = 8.84 g. Yield = 79%.

121.1 (2C), 147.4 (2C).

5-bromo-2,2-dimethylbenzo[d][1,3]dioxole (13)

12 (14.64 g, 97.6 mmol) was dissolved in DMF (98 mL), and

NBS (17.40 g, 97.7 mmol) was added. The reaction mixture was

stirred for 1 day, diluted with water (~1L) and extracted with

hexane (3x100 mL). The combined organic layers were dried

¹H NMR: (DMSO-d

Hz, 1 H), 7.24 (dd, J=8.4, 1.7 Hz, 1 H), 7.39 (d, J=1.7 Hz, 1 H),

0.60 (s, 1 H), 12.37 - 14.87 (br. s, 1 H).

, 400 MHz) δ = 5.99 (s, 2 H), 6.87 (d, J=8.4

2 4

over Na SO and evaporated. The residue was distilled at

reduced pressure (bp=80 ºC, 1 torr). M = 10.35 g. Yield = 46%.

¹³C NMR (DMSO-d

, 100 MHz): δ = 101.2, 102.2, 108.0, 113.5,

132.0, 144.0, 147.1, 156.5, 162.2.

1_H_N_M_R₍_C_D_C_l

, 400 MHz) δ = 1.68 (s, 6 H), 6.60 (d, J=8.2 Hz,

H), 6.87 (d, J=2.0 Hz, 1 H), 6.90 (dd, J=8.2, 1.8 Hz, 1 H).

General procedure for cross-coupling reaction and Boc

cleavage:

Aryl bromide (1 equiv) was dissolved in THF (1 M), Boc-pyrrole

boronic acid (1.2 equiv) was added to the solution followed by

¹³C NMR (CDCl

, 100 MHz) δ = 25.9 (2C), 109.5, 112.2, 112.5,

19.1, 123.8, 146.9, 148.5.

2-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)-1H-pyrrole (14)

aqueous Na

of nitrogen Pd(PPh

(2 equiv) solution (1 M). Under a constant flow

Cl (1 mol %) was added. The reaction

Compound 14 was prepared according to the general procedure

for cross-coupling reaction and Boc cleavage from 13. M = 2.46

g. Yield = 67% (over two steps).

)

mixture was vigorously stirred and heated under reflux for 4-5

hours. The reaction mixture was cooled to room temperature

diluted with water and extracted with DCM (3x100 mL). The

1_H_N_M_R₍_C_D_C_l

, 400 MHz) δ = 1.70 (s, 6 H), 6.27 (dd, J=5.7, 2.7

2 4

organic layer was dried over Na SO and evaporated. To the

Hz, 1 H), 6.35 - 6.39 (m, 1 H), 6.74 (d, J=8.6 Hz, 1 H), 6.79 -

.83 (m, 1 H), 6.85 - 6.93 (m, 2 H), 8.33 (br. s, 1 H).

residue was added freshly prepared (from Na and MeOH)

NaOMe solution (~1M in MeOH, 2-3 equiv) and left overnight.

The reaction mixture was diluted with water and extracted with

13_C_N_M_R₍_C_D_C_l

, 100 MHz) δ = 26.0 (2C), 105.2, 108.6, 110.0,

116.9, 118.2, 118.3, 127.0, 132.5, 146.3, 148.1.

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¹H NMR: (DMSO, 400 MHz) δ = 6.01 (s, 2 H), 6.50 (dd, J=3.5,

2.6 Hz, 1 H), 6.76 (dd, J=3.6, 2.3 Hz, 1 H), 6.90 (d, J=8.1 Hz, 1

H), 7.32 (dd, J=8.1, 1.7 Hz, 1 H), 7.45 (d, J=1.5 Hz, 1 H), 11.78

(s, 1 H), 12.27 (br. s, 1H).

General procedure for aryl pyrrole trifluoroacetylation:

To the solution appropriate 2-aryl-pyrrole and pyridine (1.2

equiv) in dichloromethane (1 M) trifluoroacetic anhydride (1.2

equiv) was added dropwise with an external cold water bath

cooling. After the addition is complete, the mixture was stirred

for 1 hour, and the yellow-greenish precipitate was filtered and

washed with DCM. If no precipitation occurred, the reaction

mixture was evaporated and triturated with water. The

precipitate was filtered and recrystallized from ethanol.

¹³C NMR: (DMSO, 100 MHz) δ = 101.2, 105.7, 107.2, 108.6,

116.6, 119.0, 123.6, 126.0, 136.7, 146.5, 147.8, 162.0.

5-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-1H-pyrrole-2-

carboxylic acid (19)

Compound 19 was prepared according to the general procedure

for haloform reaction from 16. M = 389 mg. Yield = 46%.

1-(5-(benzo[d][1,3]dioxol-5-yl)-1H-pyrrol-2-yl)-2,2,2-

trifluoroethanone (15)

Compound 15 was prepared according to the general procedure

for aryl pyrrole trifluoroacetylation from 7. M = 7.14 g. Yield =

¹H NMR: (DMSO, 400 MHz) δ = 6.65 (s, 1 H), 6.79 (s, 1 H), 7.40

(d, J=8.6 Hz, 1 H), 7.69 (d, J=8.4 Hz, 1 H), 7.93 (s, 1 H), 12.02

(s, 1 H), 12.40 (br. s, 1 H).

75%.

¹H NMR: (DMSO, 400 MHz) δ = 6.06 (s, 2 H), 6.82 (s, 1 H), 6.97

¹³C NMR: (DMSO, 100 MHz) δ = 106.9, 108.3, 110.3, 116.5,

121.3, 124.7, 128.7, 131.5 (t, J = 251.9 Hz), 135.3, 141.8, 143.5,

162.0.

(

d, J=8.1 Hz, 1 H), 7.22 (d, J=1.3 Hz, 1 H), 7.50 (d, J=8.1 Hz, 1

H), 7.61 (s, 1 H), 12.68 (s, 1 H).

¹³C NMR: (DMSO, 100 MHz) δ = 101.5, 106.5, 108.7, 110.4,

5-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)-1H-pyrrole-2-

carboxylic acid (20)

Compound 20 was prepared according to the general procedure

for haloform reaction from 17. M = 1.55 g. Yield = 76%.

17.2 (q, J = 289.8 Hz), 120.9, 123.5 (q, J = 2.9 Hz), 123.8,

25.5, 143.7, 147.9, 148.1, 167.1 (q, J = 34.4 Hz).

-(5-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-1H-pyrrol-2-yl)-

,2,2-trifluoroethanone (16)

¹H NMR: (DMSO, 400 MHz) δ = 1.63 (s, 6 H), 6.43 - 6.50 (m, 1

H), 6.76 (dd, J=3.4, 2.3 Hz, 1 H), 6.80 (d, J=8.1 Hz, 1 H), 7.28

(dd, J=8.2, 1.3 Hz, 1 H), 7.35 (d, J=1.2 Hz, 1 H), 11.77 (s, 1 H),

12.22 (br. s, 1 H).

Compound 16 was prepared according to the general procedure

for aryl pyrrole trifluoroacetylation from 10. M = 7.52 g. Yield =

65%.

¹H NMR: (DMSO, 400 MHz) δ = 6.95 (dd, J=4.2, 2.4 Hz, 1 H),

¹³C NMR: (DMSO, 100 MHz) δ = 25.6 (2C), 105.6, 107.0, 108.4,

116.5, 118.2, 118.5, 123.4, 125.4, 136.9, 146.2, 147.3, 161.9.

7.23 - 7.29 (m, 1 H), 7.48 (d, J=8.4 Hz, 1 H), 7.85 (dd, J=8.5, 1.7

Hz, 1 H), 8.06 (d, J=1.5 Hz, 1 H), 12.89 (br. s, 1 H).

5-bromobenzo[c][1,2,5]thiadiazole (22)

¹³C NMR: (DMSO, 100 MHz) δ = 107.9, 110.3, 110.9, 117.2 (q,

J = 289.8 Hz), 122.9, 123.0 (q, J = 3.5 Hz), 126.3, 126.8, 131.4

Phenylendiamine 21 (12.46 g, 67.0 mmol) was added to SOCl

(44 mL, 603 mmol, 9 equiv) followed by H SO (1.93 mL, 36.2

mmol, 0.54 equiv). The reaction mixture was heated at reflux for

hours, cooled and evaporated. The residue was suspended in

water (~0.5 L), and the product was extracted with DCM (3x100

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

(

t, J = 253.8 Hz), 142.1, 143.1, 143.5, 168.0 (q, J = 289.8 Hz).

-(5-(2,2-dimethylbenzo[d][1,3]dioxol-5-yl)-1H-pyrrol-2-yl)-

,2,2-trifluoroethanone (17)

Compound 17 was prepared according to the general procedure

for aryl pyrrole trifluoroacetylation from 14. 17 was purified by

means of column chromatography, eluent hexane/EtOAc, 1:1. M

evaporated. The residue was purified using column

chromatography, eluent hexane/EtOAc, 10:1. M = 11.80 g. Yield

= 82%.

=2.46 g. Yield = 46%.

¹H NMR (CDCl

7.83 (d, J=9.2 Hz, 1 H), 8.18 (d, J=1.6 Hz, 1 H).

, 400 MHz) δ = 7.62 (dd, J=9.2, 1.7 Hz 1 H),

¹H NMR: (DMSO, 400 MHz) δ = 1.65 (s, 6 H), 6.80 (dd, J=3.9,

2.3 Hz, 1 H), 6.89 (d, J=8.2 Hz, 1 H), 7.17 - 7.26 (m, 1 H), 7.47

(

dd, J=8.2, 1.2 Hz, 1 H), 7.51 (s, 1 H), 12.67 (s, 1 H).

¹³C NMR (CDCl

, 100 MHz) δ = 122.2, 123.9, 124.6, 133.2,

153.4, 155.3.

¹³C NMR: (DMSO, 100 MHz) δ = 25.5 (2C), 106.4, 108.6, 110.4,

17.3 (q, J = 289.8 Hz), 118.8, 120.6, 123.4, 123.6 (q, J = 2.9

5-(1H-pyrrol-2-yl)benzo[c][1,2,5]thiadiazole (24)

Hz), 125.5, 144.1, 147.5, 147.8, 167.0 (q, J = 34.4 Hz).

Compound 24 was prepared according to the general procedure

for cross-coupling reaction and Boc cleavage from 22. The

substance was triturated with heхane/EtOAc 10:1. M = 7.27 g.

General procedure for haloform reaction:

The solution of NaOH (3 equiv) in water-ethanol mixture (0.33 M, Yield = 72% (over two steps).

1:1) was added 2,2,2-trifluoroethanone (1 equiv). The resulting

reaction mixture was refluxed for 12 hours and cooled to room

temperature. Concentrated aqueous HCl solution (~12 M, 3

equiv) was added dropwise. The resulting precipitate is filtered

off and washed with water. If no precipitation occurs, the

¹H NMR: (DMSO, 400 MHz) δ = 6.20 (s, 1 H), 6.84 (d, J=2.4 Hz,

1 H), 7.02 (s, 1 H), 8.00 (d, J=9.2 Hz, 1 H), 8.08 (d, J=9.2 Hz, 1

H), 8.21 (s, 1 H), 11.65 (br. s, 1 H).

reaction mixture was extracted with Et

The combined organic layer was washed with brine, dried over

MgSO and evaporated. If necessary, the acids can be purified

using chromatography. Eluent: hexanes/EtOAc, 1:1.

O or EtOAc (3x100 mL).

¹³C NMR: (DMSO, 100 MHz) δ = 109.2, 109.9, 111.3, 121.2,

121.8, 128.7, 129.6, 134.0, 153.1, 155.4.

1-(5-(benzo[c][1,2,5]thiadiazol-5-yl)-1H-pyrrol-2-yl)-2,2,2-

trifluoroethanone (25)

Compound 25 was prepared according to the general procedure

for aryl pyrrole trifluoroacetylation from 24. M = 8.20 g. Yield =

76%.

5-(benzo[d][1,3]dioxol-5-yl)-1H-pyrrole-2-carboxylic acid (18)

Compound 18 was prepared according to the general procedure

for haloform reaction from 15. M = 5.24 g. Yield = 90%.

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10.1002/cmdc.201800534

¹H NMR: (DMSO, 400 MHz) δ = 7.12 (s, 1 H), 7.26 (s, 1 H), 8.05

water (3 mL, ~3 equiv). The resulted suspension was stirred for

10 minutes and evaporated. Chromatography with 10:1

hexanes/EtOAc, then pure EtOAc eluent gave an oil.

M = 10.67 g. Yield = 66% (S-isomer).

(

d, J=9.2 Hz, 1 H), 8.21 (d, J=9.2 Hz, 1 H), 8.72 (s, 1 H), 13.06

s, 1 H).

¹³C NMR: (DMSO, 100 MHz) δ = 112.6, 115.5, 117.0 (q, J =

289.1 Hz), 117.6, 118.4, 121.4, 123.0, 126.9, 128.6, 130.9,

141.4, 154.0, 154.5, 167.9, 168.1 (q, J = 35.3 Hz).

M = 10.53 g. Yield = 66% (R-isomer).

¹H NMR (CDCl

H), 4.45 - 4.51 (m, 2H), 5.16 (dd, J=10.5, 1.1 Hz, 1H), 5.25 (dd,

, 400 MHz): δ = 1.17 (s, 9H), 1.47 (d, J=2.4 Hz,

5-(benzo[c][1,2,5]thiadiazol-5-yl)-1H-pyrrole-2-carboxylic

J=17.2, 1.4 Hz, 1H), 5.63 (br. s, 1H), 5.79 - 5.93 (m, 1 H), 7.88

(s, 1 H).

acid (26)

Compound 26 was prepared according to the general procedure

for haloform reaction from 25. M = 6.75 g. Yield = 95%.

¹³C NMR (CDCl

57.2, 65.4, 117.8, 132.8, 154.6, 171.3.

, 100 MHz): δ = 22.4 (3C), 24.7, 24.9, 56.5,

¹H NMR: (DMSO, 400 MHz) δ = 6.86 (s, 1 H), 6.93 (s, 1 H), 8.04

(

d, J=9.3 Hz, 1 H), 8.20 (d, J=9.2 Hz, 1 H), 8.62 (s, 1 H), 12.46

br. s., 1 H), 12.31 (s, 1 H).

allyl

(1-(5-(((tert-butyldimethylsilyl)oxy)methyl)-4-

methylthiazol-2-yl)-1-(1,1-dimethylethylsulfinamido)-2-

methylpropan-2-yl)carbamate (32)

Thiazole 31 (23.66 g 97.4 mmol, 2.5 equiv) was dissolved in

THF (97 mL) and cooled to −78 °C. At this temperature, n-BuLi

¹³C NMR: (DMSO, 100 MHz) δ = 110.4, 114.9, 116.5, 121.2,

26.0, 128.9, 132.6, 134.7, 153.5, 155.0, 161.7.

(

2.5 M, 39 mL, 97.5 mmol, 2.5 equiv) was added dropwise under

allyl (1-hydroxy-2-methylpropan-2-yl)carbamate (28)

-amino-2-methylpropan-1-ol 27 (46 mL, 482 mmol, 1.5 equiv)

the nitrogen atmosphere. The reaction mixture was stirred for 30

min at −78 °C, and (enantiomer 2)-30 (10.67 g, 38.9 mmol) was

added dropwise as a solution in THF (40 mL). The reaction

mixture was slowly (∼1 h) warmed to 0 °C, and poured into

was dissolved in dichloromethane (470 mL), and water (470 mL)

and sodium bicarbonate (80.6 g, 960 mmol, 3 equiv) were then

added. To this vigorously stirred solution, allyl chloroformate (34

mL, 320 mmol) was added dropwise, and the mixture was stirred

at room temperature for 16 hours. The organic layer was

separated, and the aqueous layer was extracted with

dichloromethane (100x2 mL), the extract was dried over

anhydrous sodium sulfate. After filtration, the filtrate was

concentrated to give the pure compound. M = 47.93 g. Yield =

water (200 mL). The biphasic mixture was extracted with CH

(3×100 mL). The combined organic phases were dried over

anhydrous Na SO and evaporated. The residue was purified by

2 2

column chromatography. Eluent, hexanes/EtOAc 10:1, 3:1, 1:1

then pure EtOAc. M = 12.07 g. Yield = 60%. Compounds that

were prepared from S-30 were designated as A, compounds

that were prepared from R-30 were designated as B. 32B was

prepared using the same conditions from (enantiomer 1)-30. M =

87%.

12.11 g. Yield = 61%.

¹H NMR (CDCl

, 400 MHz): δ = 1.29 (s, 6H), 3.39 (br. s, 1H),

¹H NMR (CDCl

3.61 (s, 2H), 4.53 (d, J=5.4 Hz, 2H), 4.93 (br. s, 1H), 5.23 (dd,

J=10.5, 1.0 Hz, 1H), 5.31 (dd, J=17.2, 1.6 Hz, 1H), 5.92 (dddd,

J=16.8, 10.9, 5.7, 5.4 Hz, 1H).

, 400 MHz): δ = 0.09 (s, 6H), 0.83 - 0.95 (m, 9H),

1.23 (s, 9H), 1.32 (s, 3H), 1.42 (s, 3H), 1.54 (s, 3H), 2.33 (s, 1H),

4.47 - 4.63 (m, 2H), 4.78 (s, 2H), 5.11 (s, 1H), 5.21 (dd, J=10.3,

1.1 Hz, 1H), 5.30 (dd, J=17.2, 1.6 Hz, 1H), 5.77 (br. s, 1H), 5.85

¹³C NMR (CDCl

17.7, 132.8, 156.0.

, 100 MHz): δ = 24.3 (2C), 54.4, 65.4, 70.1,

- 5.97 (m, 1H).

allyl (1-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)-2-

methylpropan-2-yl)carbamate (33)

allyl (2-methyl-1-oxopropan-2-yl)carbamate (29)

A solution of oxalyl chloride (35 mL, 0.413 mol, 1.5 equiv) in

The 1 M HCl-MeOH solution was prepared by dropwise addition

of AcCl (7.10 mL, 0.10 mol) to methanol (100 mL) with cooling

on a water bath. The resulting solution was cooled to ambient

temperature and added to the flask containing 32A (12.07 g,

23.3 mmol). After the dissolution, the reaction mixture was

mL, 0.690 mol, 2.5 equiv) in CH

The resulting solution was stirred for 10 minutes, and a solution

of alcohol 28 (47.93 g, 277 mmol) in CH Cl (415 mL) was

added dropwise at the same temperature. After 15 minutes Et

193 mL, 1.385 mol, 5.0 equiv) was added, and 5 minutes later

(100 mL) was added dropwise to a solution of DMSO (49

Cl (415 mL) at – 70 to – 80 °C.

stirred for 1 h, evaporated, dissolved in CH

washed with 10% aqueous K CO solution (100 mL). The layers

were separated, and the aqueous layer was extracted with

CH Cl (2×100 mL). The combined organic phases were dried

over anhydrous Na SO and evaporated. The residue was

2 2

Cl (100 mL), and

(

the reaction mixture was allowed to warm to r.t. The reaction

mixture was quenched with water (1 L), and the layers were

separated. The organic layer was dried over Na

evaporated. The residue was used without purification. M =

7.40 g. Yield = 100 %.

and

separated by column chromatography. Eluent CH

(50:1, 10:1). M = 5.28 g. Yield = 76%.

2 2

Cl /MeOH

The second enantiomer (33B) was prepared according to the

same procedure from 32B. M = 4.11 g. Yield = 59%.

¹H NMR (CDCl

, 400 MHz): δ = 1.39 (s, 6H), 4.56 (d, J=5.4 Hz,

2H), 5.23 (dd, J=10.2, 1.0 Hz, 1H), 5.31 (d, J=17.2 Hz, 1H), 5.41

(br. s, 1 H), 5.91 (dddd, J=16.8, 11.0, 5.6, 5.4 Hz, 1H), 9.44 (s,

¹H NMR (CDCl

(br. s, 3 H), 4.35 (s, 1H), 4.50 (d, J=5.3 Hz, 2H), 4.70 (s, 2H),

.18 (d, J=10.4 Hz, 1H), 5.28 (d, J=17.1 Hz, 1H), 5.72 (s, 1H),

5.90 (dddd, J=16.8, 11.0, 5.6, 5.4 Hz, 1H).

, 400 MHz): δ = 1.33 (d, 6 H), 2.32 (s, 3 H), 2.76

1H).

¹³C NMR (CDCl

, 100 MHz): δ = 21.8 (2C), 59.5, 65.7, 118.0,

32.6, 155.2, 200.6.

13_C_N_M_R₍_C_D_C_l

, 100 MHz): δ = 15.1, 23.7, 23.9, 56.0, 56.3,

(E)-allyl

(1-((tert-butylsulfinyl)imino)-2-methylpropan-2-

60.1, 65.2, 117.6, 131.6, 133.0, 148.4, 155.3, 169.4.

yl)carbamate (30)

To a solution of aldehyde 29 (10.00 g, 58.5 mmol) in CH

Allyl (1-(5-(((tert-butyldimethylsilyl)oxy)methyl)thiazol-2-yl)-

1-(1,1-dimethylethylsulfinamido)-2-methylpropan-2-

yl)carbamate (35)

Thiazole 34 (36.30 g, 158.5 mmol, 2.5 equiv) was dissolved in

THF (158 mL) and cooled to −78 °C. At this temperature, n-BuLi

(2.5 M, 63 mL, 157.5 mmol, 2.5 equiv) was added dropwise

(

100 mL), R or S tert-butylsulfonamide (7.80 g, 64.4 mmol, 1.1

equiv) was added in one portion, followed by the addition of

Ti(OiPr) (17.3 mL, 58.4 mmol, 1 equiv). The resulting solution

was stirred overnight (TLC indicated consumption of the SM),

and silica (50 mL) was added to the reaction mixture followed by

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10.1002/cmdc.201800534

under the nitrogen atmosphere. The reaction mixture was stirred

for 10 min at −78 °C, and (enantiomer 2)-30 (17.35 g, 63.3

mmol) was added dropwise as a solution in THF (63 mL). The

reaction mixture was slowly (∼1 h) warmed to 0 °C, and poured

into water (0.5 L). The biphasic mixture was extracted with

layer was separated, and the aqueous layer was extracted with

DCM/EtOH (∼4:1, (2−4) × 50 mL). The combined organic layers

were dried over Na

by flash chromatography afforded amine as a slightly brown or

yellowish solid. Eluent CHCl /MeOH saturated with NH (10:1).

2 4

SO , filtered, and concentrated. Purification

over anhydrous Na

purified by column chromatography. Eluent, hexanes/EtOAc

(3×100 mL). The combined organic phases were dried

When yields are listed the first one is for compounds prepared

from ‘A’ amine, the second one is for compounds prepared from

‘B’ amine.

SO and evaporated. The residue was

0:1, then pure EtOAc. M = 21.45 g. Yield = 67%.

5B was prepared using the same conditions from (enantiomer

)-30. M = 20.08 g. Yield = 66%.

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

5-(benzo[d][1,3]dioxol-5-yl)-1H-pyrrole-2-carboxamide (NBD-

14110 and NBD-14111)

Compounds NBD-14110 and NBD-14111 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 37 and acid 18.

5 was obtained as an inseparable mixture of diastereomers

(dr~3:1).

¹H NMR: (CDCl

, 400 MHz) δ = 0.07 (s, 6 H), 0.88 (s, 9 H), 1.21

(

s, 6 H), 1.30 (s, 3 H), 1.40 (s, 3 H), 1.52 (s, 3 H), 4.45 - 4.59 (m,

NBD-14110: M = 406 mg. Yield = 35% (over two steps).

H), 4.80 - 4.86 (m, 3 H), 5.10 (s, 1 H), 5.18 (dd, J=10.5, 1.0 Hz, rt = 1.108 min. Purity = 100%. LC–MS: m/z [M+H] = 401 Da.

H), 5.28 (dd, J=17.2, 1.4 Hz, 1 H), 5.66 - 5.95 (m, 2 H), 7.51 (s, NBD-14111: M = 212 mg. Yield = 28% (over two steps).

H).

rt = 1.074 min. Purity = 100%. LC–MS: m/z [M+H] = 401 Da.

m.p. = 110-115°C.

¹³C NMR (CDCl

(3C), 25.8, 25.9, 56.5, 56.5, 58.5, 65.6, 117.8, 132.8, 138.8,

40.3, 156.1, 170.8.

, 100 MHz): δ = -5.2, 22.7, 22.8 (3C), 25.4, 25.8

¹H NMR: (DMSO, 400 MHz) δ = 1.69 (br. s, 2 H), 2.25 (s, 3 H),

2.96 (dd, J=13.3, 8.1 Hz, 1 H), 3.08 (dd, J=13.3, 5.2 Hz, 1 H),

4.52 (s, 2 H), 5.12 (dd, J=13.2, 7.6 Hz, 1 H), 5.36 (br. s, 1 H),

Аllyl

(1-amino-1-(5-(hydroxymethyl)thiazol-2-yl)-2-

6.01 (s, 2 H), 6.49 (d, J=3.5 Hz, 1 H), 6.91 (d, J=8.2 Hz, 1 H),

6.93 (d, J=3.8 Hz, 1 H), 7.29 (dd, J=8.1, 1.0 Hz, 1 H), 7.42 (s, 1

H), 8.42 (d, J=7.9 Hz, 1 H), 11.58 (br. s, 1 H).

methylpropan-2-yl)carbamate (36)

The 1 M HCl-MeOH solution was prepared by dropwise addition

of AcCl to methanol with cooling on a water bath. The resulting

solution (~200 mL) was cooled to an ambient temperature and

added to the flask containing 35A (21.45 g, 42.6 mmol). After

the dissolution, the reaction mixture was stirred for 1 h,

¹³C NMR: (DMSO, 100 MHz) δ = 14.8, 45.7, 54.3, 55.0, 101.0,

105.4, 106.4, 108.5, 112.9, 118.4, 126.2, 126.5, 132.4, 134.9,

146.1, 146.9, 147.6, 160.4, 169.4

(200 mL), and washed with

21 4 4

HRMS (ESI): m/z calcd for C19H N O

S [M+H]⁺401.1278,

evaporated, dissolved in CH

0% aqueous K CO solution (200 mL). The layers were

separated, and the aqueous layer was extracted with CH Cl

(2×100 mL). The combined organic phases were dried over

anhydrous Na SO and evaporated. The residue was separated

found 401.1278.

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

methylpropyl)-5-(benzo[d][1,3]dioxol-5-yl)-1H-pyrrole-2-

carboxamide (NBD-14117 and NBD-14116)

by column chromatography. Eluent CH

M = 7.18 g. Yield = 59%.

2 2

Cl /MeOH (50:1, 10:1).

Compounds NBD-14117 and NBD-14116 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 33 and acid 18.

NBD-14117: M = 502 mg. Yield = 58% (over two steps).

The second enantiomer (36B) was prepared according to the

same procedure from 35B. M = 8.02 g. Yield = 71%.

¹H NMR: (CDCl

rt = 1.260 min. Purity = 100%. LC–MS: m/z [M+H] = 429 Da.

, 400 MHz) δ = 1.29 (s, 6 H), 2.73 (br. s, 3 H),

4.41 (s, 1 H), 4.47 (d, J=5.0 Hz, 2 H), 4.72 (s, 2 H), 5.15 (d,

J=10.5 Hz, 1 H), 5.25 (d, J=17.1 Hz, 1 H), 5.66 (s, 1 H), 5.78 -

NBD-14116: M = 494 mg. Yield = 60% (over two steps).

rt = 1.247 min. Purity = 100%. LC–MS: m/z [M+H] = 429 Da.

5.93 (m, J=16.8, 11.0, 5.5, 5.5 Hz, 1 H), 7.44 (s, 1 H).

mp= 115-120°C (decomp.).

¹³C NMR (CDCl

, 100 MHz): δ = 23.6, 23.8, 56.1, 57.0, 59.9,

5.2, 117.6, 132.9, 139.2, 139.4, 155.2, 172.4.

¹H NMR: (DMSO-d

, 400 MHz) δ = 1.07 (s, 3 H), 1.11 (s, 3 H),

1.98 (br. s, 2 H), 2.27 (s, 3 H), 4.54 (d, J=3.4 Hz, 2 H), 5.18 (d,

J=8.9 Hz, 1 H), 5.34 - 5.43 (m, 1 H), 6.02 (s, 2 H), 6.49 (d, J=3.5

Hz, 1 H), 6.92 (d, J=8.1 Hz, 1 H), 6.95 (d, J=3.5 Hz, 1 H), 7.27

General procedure for amide coupling:

DIPEA (1 equiv) was added to an appropriate acid (3, 18, 19, 20, (dd, J=8.1, 1.7 Hz, 1 H), 7.40 (d, J=1.6 Hz, 1 H), 8.07 (d, J=9.0

6; 1 equiv) followed by DMF (10 mL per 1 g of acid) and then Hz, 1 H), 11.62 (br. s, 1 H).

HBTU (1 equiv). The resulting solution was stirred for 5 min and

added to a solution of appropriate amine (33, 36, 37, 39-42 1

equiv) in DMF (10 mL per 1 g of amine) in several portions. The

reaction mixture was stirred overnight; DMF was evaporated,

and the residue was dissolved in DCM (50 mL) and successively

washed with 5% aqueous NaOH and 10% tartaric acid or citric

acid aqueous solutions (50 mL). The organic layer was dried

¹³C NMR (DMSO-d

, 100 MHz) δ = 14.9, 27.6, 28.4, 52.6, 55.0,

58.9, 101.0, 105.4, 106.4, 108.5, 113.4, 118.4, 126.2, 126.3,

132.6, 135.0, 146.2, 146.5, 147.7, 159.9, 167.5.

25 4 4

HRMS (ESI): m/z calcd for C21H N O

S [M+H]⁺429.1591,

found 429.1588.

over Na

, filtered, evaporated, and dry loaded on silica.

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

(benzo[d][1,3]dioxol-5-yl)-1H-pyrrole-2-carboxamide (NBD-

14123 and NBD-14122)

Eluting with hexanes/EtOAc (1:1, then pure EtOAc) gave the

target compounds. The products were used in the next step

without analysis.

General procedure for deprotection:

To a solution containing protected compound (5 mmol) and N,N′-

dimethyl barbituric acid (NDMBA, 15 mmol, 3 equiv) in MeOH

Compounds NBD-14123 and NBD-14122 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 39 and acid 18.

NBD-14123: M = 337 mg. Yield = 37% (over two steps).

(

50 mL), PPh

(10 mol %) was added under a nitrogen

(5 mol %). The mixture was

rt = 1.193 min. Purity = 100%. LC–MS: m/z [M+H] = 387 Da.

atmosphere followed by Pd(dba)

NBD-14122: M = 512 mg. Yield = 47% (over two steps).

stirred for 1 day under reflux. After cooling, 50 mL of DCM was

added, and the organic phase was shaken with 10% aqueous

K CO (50 mL) to remove the unreacted NDMBA. The organic

2 3

rt = 1.136 min. Purity = 100%. LC–MS: m/z [M+H] = 387 Da.

m.p. = 200-205°C.

FULL PAPER

ChemMedChem

10.1002/cmdc.201800534

¹H NMR: (DMSO, 400 MHz) δ = 1.68 (br. s, 2 H), 2.99 (dd,

J=13.1, 7.8 Hz, 1 H), 3.12 (dd, J=13.2, 5.4 Hz, 1 H), 4.59 (s, 2

H), 5.18 (dd, J=13.2, 7.7 Hz, 1 H), 5.40 - 5.50 (m, 1 H), 6.01 (s,

NBD-14144: M = 290 mg. Yield = 50% (over two steps).

rt = 1.196 min. Purity = 100%. LC–MS: m/z [M+H] = 423 Da.

m.p. = 145-150°C.

H), 6.50 (d, J=3.7 Hz, 1 H), 6.91 (d, J=8.2 Hz, 1 H), 6.94 (d,

J=3.7 Hz, 1 H), 7.25 - 7.33 (m, 1 H), 7.42 (s, 1 H), 7.53 (s, 1 H),

8.45 (d, J=7.9 Hz, 1 H), 11.58 (br. s, 1 H).

¹H NMR: (DMSO, 400 MHz) δ = 1.81 (br. s, 2 H), 2.99 (dd,

J=13.1, 7.9 Hz, 1 H), 3.12 (dd, J=13.3, 5.3 Hz, 1 H), 4.59 (s, 2

H), 5.18 (dd, J=13.1, 7.6 Hz, 1 H), 5.46 (br. s., 1 H), 6.64 (d,

J=3.8 Hz, 1 H), 6.99 (d, J=3.8 Hz, 1 H), 7.40 (d, J=8.4 Hz, 1 H),

7.53 (s, 1 H), 7.65 (dd, J=8.5, 1.7 Hz, 1 H), 7.90 (d, J=1.5 Hz, 1

H), 8.55 (d, J=7.8 Hz, 1 H), 11.83 (br. s, 1 H).

¹³C NMR: (DMSO, 100 MHz) δ = 45.6, 54.4, 55.7, 101.0, 105.4,

06.4, 108.5, 112.9, 118.4, 126.2, 126.5, 135.0, 139.0, 140.0,

46.1, 147.7, 160.5, 172.2.

HRMS (ESI): m/z calcd for C18

S [M+H]⁺387.1122,

¹³C NMR: (DMSO, 100 MHz) δ = 45.5, 54.4, 55.8, 106.5, 107.7,

110.3, 112.8, 120.8, 127.4, 128.9, 131.2 (t, J = 252.5 Hz), 133.6,

139.0, 140.0, 141.4, 143.3, 160.5, 172.0.

found 387.1126.

5-(benzo[d][1,3]dioxol-5-yl)-N-(1-(5-(hydroxymethyl)thiazol-

17 2 4 4

HRMS (ESI): m/z calcd for C18H F N O

S [M+H]⁺423.0933,

2-yl)-2-(methylamino)ethyl)-1H-pyrrole-2-carboxamide (NBD-

14139 and NBD-14140)

found 423.0935.

Compounds NBD-14139 and NBD-14140 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 40 and acid 18.

NBD-14139: M = 385 mg. Yield = 43% (over two steps).

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

dimethylbenzo[d][1,3]dioxol-5-yl)-1H-pyrrole-2-carboxamide

(NBD-14133 and NBD-14134)

rt = 0.746 min. Purity = 100%. LC–MS: m/z [M+H] = 401 Da.

Compounds NBD-14133 and NBD-14134 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 39 and acid 20.

NBD-14133: M = 360 mg. Yield = 31% (over two steps).

NBD-14140: M = 400 mg. Yield = 43% (over two steps).

rt = 0.941 min. Purity = 100%. LC–MS: m/z [M+H] = 401 Da.

m.p. = 115-120°C.

rt = 1.279 min. Purity = 97%. LC–MS: m/z [M+H] = 415 Da.

¹H NMR: (DMSO, 400 MHz) δ = 1.91 (br. s, 1 H), 2.32 (s, 3 H),

NBD-14134: M = 426 mg. Yield = 33% (over two steps).

2.99 (dd, J=12.3, 8.3 Hz, 1 H), 3.05 (dd, J=12.5, 5.6 Hz, 1 H),

rt = 1.289 min. Purity = 99%. LC–MS: m/z [M+H] = 415 Da.

4.59 (d, J=5.3 Hz, 2 H), 5.39 (td, J=7.9, 6.0 Hz, 1 H), 5.46 (t,

J=5.6 Hz, 1 H), 6.01 (s, 2 H), 6.50 (dd, J=3.4, 2.1 Hz, 1 H), 6.89

m.p. = 100-105°C (decomp.).

6.94 (m, 2 H), 7.29 (dd, J=8.1, 1.5 Hz, 1 H), 7.42 (d, J=1.5 Hz,

H), 7.53 (s, 1 H), 8.47 (d, J=8.1 Hz, 1 H), 11.59 (s, 1 H).

¹H NMR: (DMSO, 400 MHz) δ = 1.64 (s, 6 H), 2.99 (dd, J=13.1,

7.8 Hz, 1 H), 3.12 (dd, J=13.2, 5.3 Hz, 1 H), 4.60 (s, 2 H), 5.18

(

dd, J=13.0, 7.6 Hz, 1 H), 5.48 (br. s., 1 H), 6.47 (d, J=3.8 Hz, 1

¹³C NMR: (DMSO, 100 MHz) δ = 35.5, 50.3, 54.5, 55.8, 101.0,

05.4, 106.5, 108.5, 113.0, 118.4, 126.2, 126.4, 135.0, 138.9,

H), 6.82 (d, J=8.1 Hz, 1 H), 6.95 (d, J=3.7 Hz, 1 H), 7.24 (dd,

J=8.2, 1.6 Hz, 1 H), 7.31 (d, J=1.5 Hz, 1 H), 7.54 (s, 1 H), 8.46

(d, J=7.8 Hz, 1 H), 11.56 (br. s, 1 H). Two exchangeable protons

are missing

140.1, 146.2, 147.7, 160.3, 172.2.

HRMS (ESI): m/z calcd for C19

found 401.1283.

21 4 4

H N O

S [M+H]⁺401.1278,

¹³C NMR: (DMSO, 100 MHz) δ = 25.6 (2C), 45.7, 54.4, 55.8,

105.3, 106.3, 108.3, 113.0, 118.05, 118.08, 125.7, 126.4, 135.3,

139.0, 140.0, 145.9, 147.3, 160.5, 172.2.

N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)-2-

methylpropyl)-5-(benzo[d][1,3]dioxol-5-yl)-1H-pyrrole-2-

carboxamide (NBD-14119 and NBD-14118)

23 4 4

HRMS (ESI): m/z calcd for C20H N O

S [M+H]⁺415.1435,

Compounds NBD-14119 and NBD-14118 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 36 and acid 18.

NBD-14119: M = 331 mg. Yield = 34% (over two steps).

found 415.1440.

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

(benzo[d][1,3]dioxol-5-yl)-1H-pyrrole-2-carboxamide (NBD-

14172 and NBD-14173)

rt = 1.119 min. Purity = 98%. LC–MS: m/z [M+H] = 415 Da.

NBD-14118: M = 475 mg. Yield = 53% (over two steps).

Compounds NBD-14172 and NBD-14173 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 41 and acid 18.

NBD-14172: M = 323 mg. Yield = 32% (over two steps).

rt = 1.121 min. Purity = 96%. LC–MS: m/z [M+H] = 415 Da.

mp= 105-110°C (decomp.).

¹H NMR: (DMSO-d

.01 (br. s, 2 H), 4.62 (s, 2 H), 5.24 (d, J=8.8 Hz, 1 H), 5.48 (br.

s, 1 H), 6.02 (s, 2 H), 6.49 (d, J=3.8 Hz, 1 H), 6.92 (d, J=8.1 Hz,

H), 6.95 (d, J=3.7 Hz, 1 H), 7.28 (dd, J=8.1, 1.7 Hz, 1 H), 7.40

, 400 MHz) δ = 1.09 (s, 3 H), 1.12 (s, 3 H),

rt = 1.174 min. Purity = 100%. LC–MS: m/z [M+H] = 387 Da.

NBD-14173: M = 503 mg. Yield = 51% (over two steps).

rt = 1.136 min. Purity = 100%. LC–MS: m/z [M+H] = 387 Da.

m.p. = 100-105°C.

(

d, J=1.7 Hz, 1 H), 7.56 (s, 1 H), 8.10 (d, J=9.0 Hz, 1 H), 11.62

br. s, 1 H).

¹H NMR: (DMSO-d

, 400 MHz) δ = 1.73 (br. s, 2 H), 2.98 (dd,

J=13.1, 7.8 Hz, 1 H), 3.12 (dd, J=13.2, 5.3 Hz, 1 H), 4.52 (d,

J=3.2 Hz, 2 H), 5.14 - 5.23 (m, 1 H), 5.29 (t, J=5.1 Hz, 1 H), 6.01

(s, 2 H), 6.50 (d, J=3.7 Hz, 1 H), 6.91 (d, J=8.2 Hz, 1 H), 6.94 (d,

J=3.7 Hz, 1 H), 7.27 (s, 1 H), 7.29 (dd, J=8.2, 1.7 Hz, 1 H), 7.42

(d, J=1.6 Hz, 1 H), 8.47 (d, J=8.1 Hz, 1 H), 11.60 (br. s, 1 H).

¹³C NMR (DMSO-d

, 100 MHz) δ = 27.5, 28.5, 52.7, 55.8, 59.2,

01.0, 105.4, 106.5, 108.6, 113.5, 118.5, 126.2, 126.3, 135.1,

38.7, 140.2, 146.2, 147.7, 160.0, 170.3.

HRMS (ESI): m/z calcd for C20

S [M+H]⁺415.1435,

found 415.1439.

13_C_N_M_R₍_D_M_S_O_-_d

, 100 MHz): δ = 45.8, 54.3, 59.8, 101.0,

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

difluorobenzo[d][1,3]dioxol-5-yl)-1H-pyrrole-2-carboxamide

105.4, 106.5, 108.5, 113.0, 114.0, 118.4, 126.2, 126.5, 135.0,

146.2, 147.7, 157.6, 160.5, 172.5.

19 4 4

HRMS (ESI): m/z calcd for C18H N O

S [M+H]⁺387.1122,

(

NBD-14145 and NBD-14144)

Compounds NBD-14145 and NBD-14144 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 39 and acid 19.

NBD-14145: M = 124 mg. Yield = 52% (over two steps).

found 387.1121.

5-(benzo[d][1,3]dioxol-5-yl)-N-(1-(4-(hydroxymethyl)thiazol-

2-yl)-2-(methylamino)ethyl)-1H-pyrrole-2-carboxamide (NBD-

14159 and NBD-14158)

rt = 1.143 min. Purity = 100%. LC–MS: m/z [M+H] = 423 Da.

FULL PAPER

ChemMedChem

10.1002/cmdc.201800534

Compounds NBD-14159 and NBD-14158 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 42 and acid 18.

NBD-14159: M = 611 mg. Yield = 39% (over two steps).

Cells and viruses

MT-2 cells ^[¹⁶^], TZM-bl cells ^[¹⁷^]and U87-CD4+-CXCR5+ cells

and U87-CD4+-CCR5+ cells ^[¹⁸^]were obtained through the NIH

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

negative Cf2Th-CCR5+ cells and Env expression vector

pSVIIIenv-ADA were kindly provided by Dr. J. G. Sodroski ^[¹⁹^].

The human PBMC (Peripheral blood mononuclear cells) were

isolated from buffy coats of healthy HIV-1 negative donor

obtained from the New York Blood Center (New York, NY) and

grown in RPMI 1640 medium supplemented with fetal bovine

serum (FBS) penicillin and streptomycin. The PBMC were

stimulated with 5 mg/mL phytohemagglutinin (PHA) and 20

U/mL interleukin 2 (IL-2). HIV-1 Env molecular clone expression

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

rt = 1.071 min. Purity = 100%. LC–MS: m/z [M+H] = 401 Da.

NBD-14158: M = 769 mg. Yield = 55% (over two steps).

rt = 1.056 min. Purity = 100%. LC–MS: m/z [M+H] = 401 Da.

m.p. = 150-155°C.

¹H NMR: (DMSO-d

H), 3.01 (dd, J=12.5, 8.4 Hz, 1 H), 3.08 (dd, J=12.5, 5.4 Hz, 1 H),

.54 (s, 2 H), 5.29 (br. s, 1 H), 5.43 (td, J=8.1, 5.6 Hz, 1 H), 6.01

, 400 MHz) δ = 1.90 (br. s, 1 H), 2.33 (s, 3

(s, 2 H), 6.50 (d, J=3.5 Hz, 1 H), 6.91 (d, J=8.2 Hz, 1 H), 6.94 (d,

J=3.5 Hz, 1 H), 7.26 - 7.31 (m, 2 H), 7.42 (d, J=1.7 Hz, 1 H),

8.48 (d, J=8.2 Hz, 1 H), 11.59 (br. s, 1 H).

¹³C NMR (DMSO-d

, 100 MHz) δ = 35.5, 50.3, 54.7, 59.8, 101.0,

05.4, 106.5, 108.5, 113.1, 114.1, 118.4, 126.2, 126.4, 135.1,

46.2, 147.7, 157.6, 160.3, 172.5.

S [M+H]⁺401.1278,

[20]

HRMS (ESI): m/z calcd for C19

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

pseudovirus production were obtained as follows: clones

representing the standard panels A, A1/D, A2/D,

found 401.1278.

N1-(2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-

yl)ethyl)-N2-(benzo[d][1,3]dioxol-5-yl)oxalamide (NBD-14108

and NBD-14109)

(QB099.391M.Env.C8) and D were obtained through the NIH

ARP from Dr. J. Overbaugh ^[²¹^]. The HIV-1 Env molecular clones

of subtype A/G were obtained through the NIH ARP, from Drs. D.

Ellenberger, B. Li, M. Callahan and S. Butera ^[²²^]. The HIV-1 Env

panel of standard reference subtype B Env clones were

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

and M. Li (PVO, clone 4 (SVPB11), TRO, clone 11 (SVPB12)

and QH0692, clone 42 (SVPB6)); from Drs. B. H. Hahn and J. F.

Salazar-Gonzalez (pREJO4541, clone 67 (SVPB16) and

Compounds NBD-14108 and NBD-14109 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 37 and acid 3.

NBD-14108: M = 106 mg. Yield = 15% (over two steps).

rt = 0.950 min. Purity = 100%. LC–MS: m/z [M+H] = 379 Da.

NBD-14109: M = 151 mg. Yield = 21% (over two steps).

rt = 0.947 min. Purity = 100%. LC–MS: m/z [M+H] = 379 Da.

m.p. = 155-160°C.

pRHPA4259, clone 7 (SVPB14)); from Drs. B. H. Hahn and D. L.

_K_o_t_h_e₍_p_T_H_R_O₄₁₅₆_c_l_o_n_e₁₈₍_S_V_P_B₁₅₎₎[23]. The subtype B

¹H NMR: (DMSO, 400 MHz) δ = 1.75 (br. s, 2 H), 2.24 (s, 3 H),

3.04 (d, J=6.1 Hz, 2 H), 4.52 (d, J=4.3 Hz, 2 H), 4.98 (t, J=5.8 Hz,

clones

pWEAUd15.410.5017,

p1054.TC4.1499

and

H), 5.40 (t, J=4.9 Hz, 1 H), 6.00 (s, 2 H), 6.89 (d, J=8.4 Hz, 1

H), 7.32 (d, J=8.6 Hz, 1 H), 7.45 (s, 1 H), 9.39 (br. s, 1 H), 10.64

br. s, 1 H).

p9021_14.B2.4571 were obtained through the NIH ARP from

_D_r_s_._B_._H_._H_a_h_n_,_B_._F_._K_e_e_l_e_a_n_d_G_._M_._S_h_a_w[24]. The subtype C

HIV-1 reference panel of Env clones were also obtained through

the NIH ARP from Drs. D. Montefiori, F. Gao, C. Williamson and

S. A. Karim (Du422.1, clone 1); from Drs. E. Hunter and C.

Derdeyn (ZM109F.PB4); from Drs. L. Morris, K. Mlisana, and D.

_M_o_n_t_e_f_i_o_r_i_,₍_C_A_P₂₁₀_.₂_.₀₀_._E₈₎[25]. The HIV-1 Subtype C Panel of

Indian gp160 Env Clones HIV-00836-2 clone 5, HIV-16936-2

clone 21, HIV-25711-2 clone 4 and HIV-225925-2 clone 22 were

(

¹³C NMR: (DMSO, 100 MHz) δ = 14.9, 45.2, 54.9, 55.0, 101.2,

102.2, 108.0, 113.6, 131.9, 132.8, 143.9, 146.9, 147.0, 157.9,

160.4, 167.4.

19 4 5

HRMS (ESI): m/z calcd for C16H N O

S [M+H]⁺379.1071,

found 379.1066.

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

(

benzo[c][1,2,5]thiadiazol-5-yl)-1H-pyrrole-2-carboxamide

NBD-14127 and NBD-14126)

obtained through the NIH ARP from Drs. R. Paranjape, S.

_K_u_l_k_a_r_n_i_a_n_d_D_._M_o_n_t_e_f_i_o_r_i[22]. The HIV-1 Env “Panel of Paired

Infant and Maternal Env Molecular Clones” of subtype A and

Compounds NBD-14127 and NBD-14126 were obtained

following the general procedure for amide coupling and then the

general procedure for deprotection from amine 39 and acid 26.

NBD-14127: M = 419 mg. Yield = 47% (over two steps).

C/D, were obtained through the NIH ARP from Dr. J. Overbaugh

[

¹⁴^]. The Env pseudotyped genes of BG505.T332N, KNH1144,

rt = 1.001 min. Purity = 100%. LC–MS: m/z [M+H] = 401 Da.

and B41 were kindly provided by Dr. J. P. Moore of the Weil

Cornell Medical College, NY.

NBD-14126: M = 475 mg. Yield = 50% (over two steps).

rt = 1.134 min. Purity = 100%. LC–MS: m/z [M+H] = 401 Da.

m.p. = 140-145°C.

¹H NMR: (DMSO, 400 MHz) δ = 1.78 (br. s, 2 H), 3.02 (dd,

J=13.1, 7.8 Hz, 1 H), 3.15 (dd, J=13.2, 5.3 Hz, 1 H), 4.60 (s, 2

H), 5.22 (dd, J=12.6, 7.0 Hz, 1 H), 5.46 (br. s, 1 H), 6.94 (d,

J=3.8 Hz, 1 H), 7.07 (d, J=3.8 Hz, 1 H), 7.55 (s, 1 H), 8.05 (d,

J=9.2 Hz, 1 H), 8.19 (d, J=9.3 Hz, 1 H), 8.57 (s, 1 H), 8.64 (d,

J=7.6 Hz, 1 H), 12.02 (br. s, 1 H).

The Env-deleted proviral backbone plasmids pSG3Δ^eⁿ^vDNA

(from Drs. J. C. Kappes and X. Wu) ^[¹⁷^,²³^b^]and pNL4-3.Luc.R-.E-

_D_N_A₍_f_r_o_m_D_r_._N_._L_a_n_d_a_u₎[26] were obtained through the NIH

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

expressing vector pVPack-VSV-G and a pFB-luc vector were

obtained from Stratagene (La Jolla, CA).

¹³C NMR: (DMSO, 100 MHz) δ = 45.6, 54.5, 55.8, 110.0, 113.1,

14.2, 121.2, 128.9, 132.9, 133.2, 139.0, 140.1, 153.5, 155.1,

60.3, 171.9.

HIV-1IIIB laboratory-adapted strain was obtained through the NIH

ARP.

HRMS (ESI): m/z calcd for C17

[M+H]⁺401.0849,

found 401.0852.

FULL PAPER

ChemMedChem

10.1002/cmdc.201800534

Pseudovirus preparation

LAI and HIV-1BaL) and primary (HIV-197USSN54^[²⁹^]) HIV-1 isolates

at 500 TCID50 in the absence or presence of graded

concentrations of compounds. 100 µl of culture media were

replaced every 3 days with fresh media. The supernatants were

collected 7 days post-infection and tested for p24 antigen by

ELISA. The percent inhibition of p24 production and IC50 values

were calculated by using the GraphPad Prism software.

Pseudoviruses capable of single cycle infection were prepared

as previously described ^[²⁷^]. Briefly, 5 x 10 HEK293T cells were

transfected with a solution containing the same amounts of an

HIV-1 Env-deleted pro-viral backbone plasmid pSG3^Δ^eⁿ^vDNA or

pNL4-3.Luc.R-.E- DNA and an HIV-1 Env-expression plasmid

with FuGENE HD (Promega). VSV-G pseudovirus was prepared

by transfecting the HEK 293T cells with a combination of the

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

expressing plasmid pVPack-GP, the pFB-luc plasmid and

FuGENE HD. Pseudovirus-containing supernatants were

collected two days after transfection, filtered, tittered and stored.

Single-cycle infection assay in U87-CD4-CCR5 cells. The

activity of the small polycyclic molecules was also tested against

control pseudovirus VSV-G, obtained as described above.

Briefly, U87-CD4-CCR5 cells were platted in a 96-well tissue

culture plate at 1 x 10⁴/ well and cultured overnight. The

following day, aliquots of VSV-G pseudovirus pre-treated with

graded concentrations of the polycyclic compounds for 30 min,

were added to the cells and incubated for 3 days. Cells were

washed and lysed with 40 µl of lysis buffer. The lysates were

then transferred to a white plate and mixed with the luciferase

assay reagent. The luciferase activity was immediately

measured to calculate the percent of inhibition and IC50 values

by using the GraphPad Prism software.

Measurement of antiviral activity

Single-cycle infection assay in TZM-bl cells. The new

generation of polycyclic NBD compounds was evaluated in

single-cycle infection assay for their anti-HIV-1 activity by

infecting TZM-bl cells with an HIV-1 pseudovirus expressing the

Env from the lab-adapted HIV-1HXB-2 (X4). Also, NBD-14113,

NBD-14110, NBD-14123, and NBD-14159 were tested against a

large number of HIV-1 pseudotyped viruses expressing the Env

Evaluation of cytotoxicity

from a panel of diverse clinical isolates as previously described

^[²⁷^].

To this end, TZM-bl cells were platted at 1x10 / well in a 96-

TZM-bl, U87-CD4-CCR5 and U87-CD4-CXCR4 cells. The

cytotoxicity of the small polycyclic molecules in these cells was

determined by using the colorimetric method CellTiter 96®

AQueous One Solution Cell Proliferation Assay (MTS)

(Promega) following the manufacturer’s instructions. Briefly, the

well tissue culture plate and cultured. Following overnight

incubation, aliquots of HIV-1 pseudoviruses were pre-treated

with graded concentrations of the small molecules for 30 min

and added to the cells. Following 3 days of incubation, the cells

were washed and lysed. 20 µl of the lysates were transferred to

a white plate and mixed with the luciferase assay reagent

cells were platted in a 96-well tissue culture plate at 1 x 10 / well

and cultured at 37 °C. Following overnight incubation, the cells

were incubated with 100 µl of the compounds at graded

concentrations and cultured for 3 days. The MTS reagent was

added to the cells and incubated for 4 h at 37 ºC. The

absorbance was recorded at 490 nm. The percent of cytotoxicity

and the CC50 (the concentration for 50 % cytotoxicity) values

were calculated as above.

(

Promega). The luciferase activity was immediately measured

with a Tecan infinite M1000 reader, and the percent inhibition by

the compounds and IC50 (the half maximal inhibitory

concentration) values were calculated by the GraphPad Prism

software.

Multi-cycle infection assay in MT-2 cells. The antiviral activity

of the small polycyclic molecules was also evaluated against the

full-length laboratory-adapted HIV-1IIIB as previously described

^[²⁸^]. In short, aliquots of HIV-1IIIB at 100 TCID50 were pre-

incubated with an equal volume of graded concentrations of

compounds for 30 min then added to the MT-2 cells at

MT-2 cells. The cytotoxicity of the NBD small molecules was

measured in MT-2 cells with the colorimetric method CellTiter

96® AQueous One Solution Cell Proliferation Assay (MTS).

Briefly, 100 µl of a small molecule at graded concentrations was

added to an equal volume of cells (10 cells / ml) in 96-well

1x10 /well in a 96-well tissue culture plate and incubated

plates. The following day, the culture supernatants were

replaced with fresh media and incubated for 4 days. The MTS

reagent was added to the cells and incubated for 4 h at 37 ºC.

The absorbance was recorded and the percent of cytotoxicity

and the CC50 values were calculated as above.

overnight. The culture supernatants were then replaced with

fresh media and cultured for 4 days. Finally, the supernatants

were collected and mixed with an equal volume of 5% Triton X-

100 and tested for p24 antigen by sandwich-ELISA. The percent

inhibition of p24 production and IC50 values were calculated by

the GraphPad Prism software.

Uninfected PBMC cells. The toxicity of the small molecules in

freshly isolated and stimulated PBMC cells was measured with

the colorimetric method CellTiter 96® AQueous One Solution

Cell Proliferation Assay. Briefly, 100 µl of escalating

concentrations of compounds was added to an equal volume of

Multi-cycle infection assay in PBMC. The inhibitory activity of

the NBD compounds on infection of PBMC by 3 HIV-1 isolates

_w_a_s_d_e_t_e_r_m_i_n_e_d_a_s_p_r_e_v_i_o_u_s_l_y_d_e_s_c_r_i_b_e_d[15]. The PHA-stimulated

cells (5 x 10 / ml) in a 96-well plate and incubated overnight.

100 µl of culture media were replaced every 3 days with fresh

PBMC (5 × 10 cells/well) were infected with lab-adapted (HIV-

FULL PAPER

ChemMedChem

10.1002/cmdc.201800534

media and incubated for a total of 7 days. The MTS reagent

was added to the cells and incubated for 4 h at 37 ºC. The

absorbance was recorded and the percent of cytotoxicity and the

CC50 values were calculated as above.

Assay in Cf2Th-CCR5 cells

CD4-negative Cf2Th-CCR5 cells were infected with the

luciferase-expressing recombinant CD4-dependent pseudovirus

[

HIV-1ADA as previously described . Briefly, the Cf2Th-CCR5

Cf2Th-CCR5 cells. The Cf2Th-CCR5 cells were platted in a 96-

cells were plated at 6×10 cells/well in a 96-well tissue culture

well tissue culture plate at 6 x 10 / well and cultured at 37 °C.

plate. Following overnight incubation, aliquots of the pseudovirus

HIV-1_A_D_Awere pre-treated with graded concentrations of the

small polycyclic molecules for 30 min then, added to the cells

and cultured for 48 hours. Cells were washed with PBS and

lysed with 40 µl of cell lysis reagent. Lysates were transferred to

a white 96-well plate and mixed with 100 µl of luciferase assay

reagent. The luciferase activity was immediately measured to

obtain the relative infection concerning the untreated control.

The Relative virus infectivity indicates the amount of infection

detected in the presence of the compounds divided by the

amount of infection detected in the absence of the compounds.

Following overnight incubation, the cells were incubated with

100 µl of the compounds at graded concentrations and cultured

for 48 h. As described above, the MTS reagent was added to the

cells and incubated for 4 h at 37 ºC. The absorbance was

recorded and the percent of cytotoxicity and the CC50 values

were calculated as above.

Single-cycle infection assay in U87-CD4-CXCR5 cells. The

activity of the small polycyclic molecules was also tested against

control pseudovirus VSV-G, obtained as described above.

Briefly, U87-CD4-CXCR5 cells were platted in a 96-well tissue

culture plate at 1 x 10⁴/ well and cultured overnight. The

following day, aliquots of VSV-G pseudovirus pre-treated with

graded concentrations of the polycyclic compounds for 30 min,

were added to the cells and incubated for 3 days. Cells were

washed and lysed with 40 µl of lysis buffer. The lysates were

then transferred to a white plate and mixed with the luciferase

assay reagent. The luciferase activity was immediately

measured to calculate the percent of inhibition and IC50 values

by using the GraphPad Prism software.

Quantitative Determination of HIV-1 Reverse Transcriptase

activity.

The polycyclic NBD compounds were also evaluated for their

activity against the HIV-1 Reverse Transcriptase (RT) by using

the Colorimetric Reverse Transcriptase Assay (Roche) and

following the manufacturer’s instructions. NBD-556 and

Nevirapine (non-nucleoside reverse transcriptase inhibitor

(

NNRTI)) were used as controls.

Evaluation of cytotoxicity

ENV-mutated pseudovirus assay

TZM-bl cells. The cytotoxicity of the small polycyclic molecules

in TZM-bl cells was determined by using the colorimetric method

CellTiter 96® AQueous One Solution Cell Proliferation Assay

We introduced the amino acid substitutions S375Y and S375W

into the pHXB2-Env expression vector by site-directed

mutagenesis (Stratagene) using mutagenic oligonucleotides as

previously described^[⁷^c^]. The ENV plasmids carrying the above

amino acid substitutions were sequenced and analyzed with the

Geneious R8 software (Biomatters, New Zealand). The

pseudoviruses were obtained by using the Env-deleted proviral

backbone plasmids pNL4-3.Luc.R-.E- DNA as described above.

U87-CD4-CXCR4 cells were infected with the ENV-mutated

pseudoviruses pretreated for 30 min with escalating

concentrations of the NBD compounds and incubated for 3 days.

Cells were washed with PBS and lysed with 40 ml of cell culture

lysis reagent. Lysates were transferred to a white 96-well plate

and mixed with 100 ml of luciferase assay reagent. We

immediately measured the luciferase activity to calculate the

IC50 as described above.

(

MTS) (Promega) following the manufacturer’s instructions.

Briefly, TZM-bl cells were platted in a 96-well tissue culture plate

at 1x10⁴/ well and cultured at 37 °C. Following overnight

incubation, the cells were incubated with 100 µl of the

compounds at graded concentrations and cultured for 3 days.

The MTS reagent was added to the cells and incubated for 4 h

at 37 ºC. The absorbance was recorded at 490 nm. The percent

of cytotoxicity and the CC50 (the concentration for 50 %

cytotoxicity) values were calculated as above.

MT-2 cells. The cytotoxicity of the NBD small molecules was

measured in MT-2 cells with the colorimetric method CellTiter

96® AQueous One Solution Cell Proliferation Assay (MTS).

Briefly, 100 µl of a small molecule at graded concentrations was

added to an equal volume of cells (10 cells/ml) in 96-well plates.

The following day, the culture supernatants were replaced with

fresh media and incubated for 4 days. Four hours after the

addition of MTS reagent the soluble intracellular formazan was

quantitated and the percent of cytotoxicity and the CC50 values

were calculated as above.

Acknowledgments

This study was supported by funds from NIH Grant R01

AI104416 (AKD), the New York Blood Center (AKD.

FULL PAPER

ChemMedChem

10.1002/cmdc.201800534

[

24]

B. F. Keele, E. E. Giorgi, J. F. Salazar-Gonzalez, J. M. Decker, K.

T. Pham, M. G. Salazar, C. Sun, T. Grayson, S. Wang, H. Li, X.

Wei, C. Jiang, J. L. Kirchherr, F. Gao, J. A. Anderson, L. H. Ping, R.

Swanstrom, G. D. Tomaras, W. A. Blattner, P. A. Goepfert, J. M.

Kilby, M. S. Saag, E. L. Delwart, M. P. Busch, M. S. Cohen, D. C.

Montefiori, B. F. Haynes, B. Gaschen, G. S. Athreya, H. Y. Lee, N.

Wood, C. Seoighe, A. S. Perelson, T. Bhattacharya, B. T. Korber,

B. H. Hahn, G. M. Shaw, Proc.Natl.Acad.Sci.U.S.A 2008, 105,

Keywords: HIV-1. ENV-pseudovirus. virus entry antagonist.

structure-activity relationship (SAR). Reverse transcriptase (RT)

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Entry for the Table of Contents

The1,3-benzodioxolyl moiety and its bioisotere, 2,1,3-benzothiadiazole were well tolerated in the Region I, which site was traditionally

known as not suitable for substitution by a bulky group. Three of the NBD inhibitors containing a 1,3-benzodioxolyl moiety showed

some improvement in the antiviral activity, but more significantly the selectivity index (SI) increased by 2-3-fold compared to the

control inhibitor NBD-14113.

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