Abstract
We present the development of alternative scaffolds and validation of their synthetic pathways as a tool for the exploration of new HIV gp120 inhibitors based on the recently discovered inhibitor of this class, NBD-14136. The new synthetic routes were based on isosteric replacements of the amine and acid precursors required for the synthesis of NBD-14136, guided by molecular modeling and chemical feasibility analysis. To ensure that these synthetic tools and new scaffolds had the potential for further exploration, we eventually tested few representative compounds from each newly designed scaffold against the gp120 inhibition assay and cell viability assays.
Keywords: HIV, gp120, drug discovery, lead optimization, synthesis, screening
Graphical Abstract
1.0. Introduction
Viruses are biological entities that are not capable of self-replicating by themselves, and they require the support of a host cell transcriptional genetic apparatus for their life cycle. Therefore, all viruses require that their genetic information (DNA or RNA, depending upon the Virus typology) reaches the host cell nucleus for replication. They have developed specific molecular mechanisms and strategies to achieve that. The blockage of these processes prevents virus proliferation and infections. This strategy has been pursued in virtually all virus pathologies, including Human Immunodeficiency Virus (HIV). Several HIV entry targets have been identified[1–3] and validated as critical players in the AIDS pathology. Among the HIV entry targets, the envelope glycoprotein, gp120, has emerged as a well-validated target[4, 5], and its inhibition is critical for bringing additional therapy to HIV infections.[6]
In 2005, we first reported identifying a small-molecule inhibitor, NBD-556, that targets HIV1 gp120[7]. However, this inhibitor was later shown to induce HIV-1 infection in CD4-CCR5+ cells, thereby acting as a gp120 agonist[8–10]. Although this was an initial setback for our endeavor to discover gp120 targeted entry inhibitors, we made continuous strides in modifying all three regions of NBD-556 (Figure 1). Most notably, we used the scaffold hopping approach to alter the oxalamide moiety to a diverse scaffold to convert NBD-556 from being a gp120 agonist to an antagonist. Our major success came when we replaced the 2,2,6,6-tetramethylpiperidine scaffold of NBD-556 with a (4-methyl-2-(piperidin-2-ylmethyl)thiazol-5-yl)methanol scaffold and obtained NBD-09027. This inhibitor showed partial agonist characteristics. However, we successfully converted the oxalamide moiety of NBD-09027 to a pyrrole and kept the other portions in Region I and Region III (Figure 1) the same as in NBD-09027. This transformation yielded NBD-11021, which showed a complete reversal of gp120 agonist to gp120 antagonist trait[8], and it was our first, second-generation inhibitor that showed potent antiviral activity (Figure 1). Therefore, the scaffold hopping approach achieved our goal of optimizing NBD-556 from a gp120 agonist to a gp120 antagonist with potent anti-HIV-1 activity. This report will concentrate on the scaffold hopping in all three regions, focusing on the region I and III.
Figure 1.
Chronological development of converting a gp120 agonist to a gp120 antagonist[7, 9, 11–16, 35] and comparison of the chemical structures of the lead gp120 inhibitors NBD-556[7], NBD-09027[15], NDB-11021[8], NBD-14136[14], and NBD-14270[11] and their antiviral activity against pseudovirus HIV-1HXB-2 (IC50), Cytotoxicity (CC50), and selectivity index (SI).
One of the recent HIV-1 gp120 entry antagonists, namely NBD-14270[11], designed in our laboratory, showed excellent antiviral profile and cytotoxicity. It was derived from a bioisosteric replacement of the aryl-pyrrole portion in Region I of its analog NBD-14136[11] (Figure 1) by introducing a pyridine ring in place of the phenyl and a methyl group on the pyrrole portion. Therefore, we hypothesize that additional isosteric replacements of this lead compound may yield inhibitors with improved antiviral potency and cytotoxicity. The majority of the NBD-based gp120 entry inhibitor arrays[2, 7, 8, 11–14] were derived from an amine and an acid portion coupled together via an amide bond, as shown in Figure 2.
Figure 2.
Schematic representation of the retrosynthetic approach of the NBD gp120 compound array and how they can be envisioned by a coupling reaction from an Aryl/heteroaryl pyrrole acid (left part) and a thiazole amine derivative (right part) and the “matrix-like” idea for generating new gp120 orientated inhibitor compounds by using bioisosteric replacements of the acids and the amine portions. PG = Protecting Group.
Albeit, the aryl portion of NBD-14136 was mostly explored, mainly via probing a different kind of aryl moieties[2, 8, 11, 13–17], yet other heteroaromatic or different aromatic systems than aryl or pyridine were not probed. Similarly, the amine portion was not entirely explored. Herewith in we report a series of new validated synthetic pathways of the left and right parts (regions I and III) that enable additional scaffold hopping attempts driven by isosteric modifications of NBD-14136 and NBD-14270.
2.0. Results and Discussion
The design of the alternative scaffolds of acids and amines to be used in the coupling reaction was done, taking into account several cycles of molecular modeling[8, 15] (data not included), a chemistry feasibility check, and corresponding reagent availabilities. The synthetic approach for each new acid and amine is reported in the chemistry section.
The antiviral activity of the NBD compounds was evaluated in a single-cycle infection assay by infecting TZM-bl cells with the HIV-1 NL4–3-HXB2-Luc pseudotyped virus expressing Env of the lab-adapted HIV-1HXB-2 (CXCR4). The cytotoxicity of the small molecules in TZM-bl cells was measured by the colorimetric XTT method, as previously described[8, 15].
2.1. Scaffold hopping of the aryl-pyrrole portion via intermediate acid replacements
In the past, we probed oxalamide-based acids, which led to compounds behaving as partial gp120 agonists activity (Figures 1 and 3)[8, 16], and one of the most active compounds obtained from the oxalamide series were NBD-09027 (Figure 1 and 3) with the para-chlorophenyl residue on the acid intermediate and the amine portion constrained in a piperidine ring. The antiviral activity of these compounds ranged in the low micromolar, but they had relatively high cytotoxicity (CC50 about 30 μM)[8, 15]. Since no single compound derived from the para-chlorophenyl oxalamide scaffold having, instead of the piperidine a methylenamine moiety as per the full agonist NBD-14091 was ever tested[7, 9, 11, 12, 15, 16], we decided to fill the gap and by exploiting promptly available intermediates from previous works (amines 1a, 2a[14] and acid 1b (Table 1), thus producing compounds 1 and 2 (as per the procedure depicted in Scheme 5) in both enantiomeric forms. On top, an additional elongated analog compound 3 (Table 1), derived from acid 3c (Scheme 1) with an extra methylene unit and a pCl-mF-benzyl residue ring, was also made following the general procedures as per Scheme 5. The rational design of compound 3 came from an attempt to improve compound 4 Sp2 character and rigidity (Figure 3). Antiviral activity (IC50) of all three pairs of enantiomers ranged in the 10–20 μM activity with problematic cytotoxicity; therefore, no further efforts were attempted in this direction (Table 1).
Figure 3.
Summary of chemical structures of the NBD-HIV-1 entry inhibitors obtained from aryl-pyrrole replacements of the lead scaffold, their IC50 against pseudovirus HIV-1HXB-2 and CC50 profiles compared to their similar known analogs, NBD-14091, NBD-14092[14] NBD-09027[15], NBD-14108[12]. The stereochemistry descriptors (fR) and (fS) were discussed in earlier work[13].
Table 1:
gp120 targeted inhibitors (products) derived from corresponding acids, and amines, and their antiviral activity (IC50) against HIV-1HXB2, HIV-1#11578 and cell viability values (CC50).
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All the starred compounds have been reported with the corresponding protecting group/s, which is not present in the final product structure since it was cleaved after that the coupling took place. The actual procedure is well described in the chemistry section for each specific case.
Intermediate synthesized and reported previously[12].
Not an acid intermediate and final product obtained via amine/isocyanate condensation instead. Details are reported in the corresponding chemistry section.
Scheme 5:
Schematic representation of the coupling and cleavage strategies adopted for compounds 1–2, 4, 6–13. R-COOH as per Table 1
Scheme 1:
Depiction of the synthetic approach to acid 3c
2.2. Pyrrole “open” form replacements
Among the alternative acid replacements, compound 4 (Figure 3), an “open” pyrrole form of a previously tested compound NBD-14091 with a 0.75 μM IC50,[14], which required starting a boc-protected acid 4b (Table 1) was commercially and promptly available, was considered. Compound 4b undergone coupling and cleavage reactions against amines 4a(fR) and 4a(fS)[14] [stereochemical descriptor f(R) and f(S) defined in the previous work[13] as per the general procedures A and B (Section 2.5.), and according to the pathway depicted in Scheme 5 yielding compounds 4(fS) and (fR)]. None of these derivatives showed any appreciable antiviral activity.
Since 4 (fR) and 4 (fS) presented a significant loss of activity compared to NBD-14091 and NBD-14092, we speculated that this could be attributed to the destruction of the rigidity Sp2 pyrrole saturated system. Therefore, we envisioned compound 3 (Figure 3), as mentioned above, and compound 5 (Scheme 2), which could still be considered as an “open form” of the pyrrole, however being more rigid due to its extended Sp2 urea-like system.
Scheme 2:
Compound 5 synthetic pathway. General procedure B: Pd(dba)2, Ph3P, MeOH
However, compounds 5 and 3, in both enantiomeric forms (fR and fS) failed to show any significant antiviral activity (Table 1 and Figure 3), and no other amines were explored against these fragments.
2.3. Phenyl ring replacement with a saturated cyclohexyl system.
A glide-base docking study indicates that the phenyl portion of NBD-14136 seems to occupy a hydrophobic region of the gp120 binding site (Figure 4a). Therefore, we envisioned its replacement with a saturated version of it by introducing a cyclohexyl ring to replace the phenyl ring on the pyrrole acid as per Scheme 3. It is noteworthy that besides these hydrophobic interactions, the salt-bridge formation of the primary amine group in these inhibitors with Asp368 is essential. In addition, the H-bond interaction of the hydroxyethyl group with Gln429 may also play an important role (Figure 4a).
Figure 4.
Glide-based docking pose of a) NBD-14136 and b) 12 9fR) bound to a hydrophobic pocket in the HIV-1 envelope glycoprotein gp120. H-bonds and ionic interactions are indicated by pink arrows, π-π interactions are indicated by green arrows.
Scheme 3:
Synthetic pathway adopted for producing acid 6c.
The resulting acid 6c was coupled, as per Scheme 5, against the enantiopure amines 3a(fS) and 3a(fR), leading respectively to the target compounds 6(fS) and 6(fR), that unfortunately were poorly actives (Table 1 and Figure 3). This leads to the speculation that the Sp2 system of the phenyl ring may play an essential role in the antiviral activity taking part in a possible a π-π interaction or any other crucial interaction.
2.4. Aryl-pyrrole replacement with 4-(cyclohexylmethyl-N/O)phenyl moiety
After considering few chemically viable replacements of the aryl-pyrrole portion, from a molecular modeling study, it emerged that 4-(cyclohexylmethoxy)phenyl and the 4-(cyclohexylmethylamino)phenyl moieties, among other considered, occupied almost the same distance of the aryl-pyrrole moiety in the gp120 pocket, at the same time keeping the same crucial key interactions. The two acids were synthesized in good yields as per the pathway depicted in Scheme 4 to verify if these kinds of replacements were fruitful.
Scheme 4:
Synthetic scheme for acid 7d and 8d
These acids, 7d and 8d, were then reacted with the enantiopure amines 3a(fR) and 3a(fS)[13], accordingly the general procedures A and B (described in the chemistry Section 2.5.) and as per the depicted in Scheme 5. The resulting products 7(fR), 7(fS), and 8(fR), 8(fS), were poorly active (Table 1). To probe if a slightly different distance between the phenyl ring and the amide moiety could have affected the activity, similar analogs of these compounds, namely 9(fR) and 9(fS) (Figure 3), with an extra methylene unit in between the phenyl and carbonyl group were also synthesized (general procedure as per Scheme 5; commercial starting acid as per Table 1), which, however, also showed no significant antiviral activity (Table 1 - Figure 3). Therefore these kinds of scaffolds were not pursued any further.
2.5. Indole as fused ring system bioisostere of the aryl-pyrrole portion
As an additional attempt to replace the phenyl-pyrrole part, we envisioned the possibility of a fused form of the phenyl and pyrrole rings, such as the indole ring. By molecular modeling such indole replacements against few previously used amines, we were able to identify three commercial indole-based acids, namely 10b, 11b, and 12b (Table 1), for which their respective final compounds had good dock scores in the Phe43 cavity of HIV-1 gp120. Therefore, their synthesis was prompted following the general procedure A and B as per Scheme 5 and lead to compounds 10, 11, and 12 in both enantiomeric forms (Figure 3). Out of them, the enantiomeric pair 12(fR) and 12(fS) reported having a reasonably good antiviral activity (5.1–6.4 μM), comparable to reference compounds NBD-14091–2 (0.75–4.3μM), however, presenting a better selectivity index with a CC50 improvement of nearly two-fold (83–84 μM versus ∼30μM for NBD-14091–2)[13]. Glide-based docking in Figure 4 (b) indicated similar interactions as were observed with NBD-14136. However, there were some notable differences, too; namely, the ethyl hydroxy group in the thiazole ring of 12 (fR) forms an H-bond with Trp427. Furthermore, Trp427 forms two π-π interactions with the phenyl pyrrole part.
The fact that 12 (both fR and fS) had moderate activity, while 10 and 11 were virtually inactive, can be attributed to its Cl in position 6. First, we created the 3D structures of 11 and 12 as described under section 3.0.2. Then, we used the Ligand Align wizard in maestro within Schrodinger Suite 2020–4 to overlap NBD-14091 with 11 and 12 as a racemate (Figure 5). It is evident how the p-Cl moiety of NBD-14091 occupies a similar place as the 6-Cl-indole moiety in 12. On the contrary, Cl in 11 points in a different position in the long but narrow active site. This result points out that the indole position 6 is more beneficial for substitution than the 5, but that a lipophilic and small substituent in the para position of the phenyl ring is crucial. Thus, rendering the corresponding indole acid 12b is expected to be a valuable tool for other gp120 inhibitor scaffold design and explorations.
Figure 5.
Comparison of the chemical structures of NBD-14091, 11, 12 and the 3D-aligned structures of 11 and 12 with NBD-14091. 12 was used as the racemate.
2.6. scaffold hopping via intermediate amine replacements of unsubstituted thiazole
While different substituted thiazole-containing compounds have previously been tested[8, 11–16], the unsubstituted thiazole ring was never considered. Therefore we made the corresponding protected unsubstituted thiazole-based amines 13c(fS) and 13c(fR) as per the synthetic route in Scheme 6 and eventually coupled them with acid 13d (Table 1) as per the procedure depicted in Scheme 5.
Scheme 6:
Schematic representation of the synthetic strategy adopted for the alloc protected amines 13c in both enantiomeric forms.
Interestingly, the resulting enantiomeric products 13(fS) and 13(fR) had a comparable safety index and antiviral activity (2.4–1.0 μM, Fig 6 and Table 1) to NBD-14091 and NBD-14092 (0.75–1.3 μM)[13], rendering this amine a useful reagent tool for further coupling reactions with acids to generate HIV-1 gp120-targeted inhibitors.
Figure 6.
Chemical structures of final compounds 13-18 with their relative antiviral activity against pseudovirus HIV-1HXB-2 (IC50) and cytotoxicity profile (CC50), in comparison with NBD-556[7].
Since the unsubstituted thiazole simplification leads to a similar antiviral activity, we decided to explore an even more drastic simplification by completely removing the thiazole ring and testing compound 14 (Figure 3), which was obtained by reacting the mono-boc-protected ethylendinamine 14a (Table 1) against acid 14b (Table 1 - acid explicitly selected since it was among one of the best-performing acids from the previous projects[13]) as per the general procedure A and B (chemistry Section 2.5.). Interestingly, 14 showed antiviral activity at low μM range for gp120 inhibition (Tab 1 and Fig 6), but at the same time demonstrated significant cytotoxicity (CC50 17.8±0.9μM).
The cytotoxicity further deteriorated when we tested a more rigid isosteric version than 14, like compounds 15 and 16 (Figure 6 for the chemical structures, Table 1 for the amine precursors, and the general coupling procedure A and B as per Section 2.5.) where the ethylenediamine portion was replaced with moieties containing piperidine ring. Both compounds had a CC50 of about 10 μM, while the antiviral activity was 4.9 μM for 15 and 13.2 μM for 16. Clearly, the cytotoxicity and the weak antiviral activity being a limitation for this kind of variations.
Nevertheless, considering that the amine’s rigidification seemed to be beneficial for the activity, despite worsening the cell viability, we envisioned the testing of a “thiazole-containing” scaffolds having the amine moiety trapped in a piperidine ring such as compounds 17 and 18 (Figure 6). Thus, compound 17 was synthesized following the route depicted from Scheme 7.
Scheme 7:
Synthetic route for amine 17i and the final product 17. General procedure A: HBTU, DIPEA, DMF. Acid 17k from previous work[17].
Achieving compound 17 was an exciting result from the merely synthetic point of view. Its chemistry is novel, and the amine 17h represents an interesting novel fragment for FBDD, being Ro3 compliant[18] on top of containing privileged drug-like elements[19]. We have calculated the drug-like properties of all inhibitors and included them in the Supplementary Materials. Moreover, its boc-protected version 17i, represent an attractive medicinal chemistry reagent since it contains at least three synthetic handles or point of diversity[20–22] that could be expanded judicially. Therefore, compounds 17h-i represent an interesting tool for any medicinal chemists for the de-novo design of new scaffolds for further synthetic manipulations.
Notably, 17 showed promising antiviral activity (5 μM) with cytotoxicity (33.7 μM) comparable to NBD-14091 and NBD-14092, however presenting the advantage of not being chiral, therefore, having a more cost-effective synthetic protocol. Thus, this scaffold, too, along with the indole variation, may represent an alternative and more economical tool for the chemical space exploration for the design of HIV-1 gp120-targeted inhibitors.
However, compounds 18(fR) and 18(fS), obtained as per the synthetic pathway represented in Scheme 8, were not attractive due to their low antiviral activity and cell viability profiles (Table 1).
Scheme 8:
Synthetic pathway of compounds 18c and 18. Acid 17k structure as per Table 1
As pointed out, the importance of the intermediary compounds 17i-h from the target compound 17, similarly compound 18c, can be seen in the same way, since it is novel, with low molecular weight (MW) (241 Da) and containing privileged drug-elements[19].
2.7. Antiviral activity of the NBD compounds against the HIV-1WEAUd15.410.5017 clinical isolate.
Antivirals may display different neutralization activity against laboratory-adapted and clinical isolates HIV-1 clones. Therefore, we decided to evaluate the activity of the NBD compounds against a clinical isolate of subtype B, HIV-1WEAUd15.410.5017 (NIH # 11578), which is a dual-tropic (CCR5/CXCR4) clinical isolate. We found that the IC50s detected against this isolate were similar to the IC50s detected against the lab-adapted HIV-1HXB-2 (Table 1). Compounds 12(fR), 12(fS), 13(fR), 13(fS), and 17 displayed the best antiviral activity against the clinical isolate confirming the findings with the lab-adapted HIV-1HXB-2. These five compounds were further investigated. At first, we tested their specificity against the control pseudovirus VSV-G (Table 2) by infecting U87-CD4-CCR5 cells as previously described[23, 24]. We found that 12(fR), 12(fS), 13(fR), 13(fS) were poorly active against VSV-G compared to the activity we detected against the two HIV-1 clones suggesting that the inhibitory activity of these compounds is specific to HIV-1, while the IC50 detected for 17 against this control virus was similar to the IC50s detected against the HIV-1 clones suggesting that 17 has no specificity for HIV-1. These compounds did not induce toxicity in the U87-CD4-CXCR5 cell line used for this assay.
Table 2.
Antiviral activity of the NBD compounds against control pseudovirus MLV-GP/VSV-G/Luc (IC50) evaluated in U87-CD4-CCR5 cells and cytotoxicity (CC50).
| Compound | U87-CD4-CCR5 cells/ VSV-G | |
| IC50 (μM)a | CC50 (μM)a | |
| 12(fR) | 15.7±1 | >86 |
| 12(fS) | 20.5±0.5 | >86 |
| 13(fR) | 12±0.8 | 45.5±1.5 |
| 13(fS) | 16.2±0.5 | 59.1±0.9 |
| 17 | 6.2±0.8 | 57.7±3.4 |
The reported IC50 and CC50 values represent the means ± standard deviations (n = 3).
2.8. Activity of the NBD compounds against HIV-1 Reverse Transcriptase (RT)
We previously reported that some early generation of NBD compounds displayed moderate activity against HIV-1 Reverse Transcriptase (RT)[12, 24]. Here, we decided to evaluate the activity of the selected five NBD compounds against that enzyme with a colorimetric RT immunoassay (Table 3). We used NBD-556 and Nevirapine as controls. We found that 12(fR) and 12(fS) alike the first-generation compound NBD-556, had no activity against RT HIV-1, while 13(fR) and 13(fS) displayed moderate RT inhibition with an IC50 of 31±2.5 and 18.9±4.7 μM, respectively; however, 17 was more effective against RT (IC50 of 1.7±0.2). In addition, nevirapine used as positive control showed potent activity against HI-1 RT with IC50 of <0.5 μM.
Table 3.
Activity of NBD Compounds against HIV-1 Reverse Transcriptase (RT)
| Compound | IC50 (μM) |
| NBD-556 (Control) | >300 |
| 12(fR) | >285 |
| 12(fS) | 245±20 |
| 13(fR) | 31±2.5 |
| 13(fS) | 18.9±4.7 |
| 17 | 1.7±0.2 |
| Nevirapine (Control) | <0.5 |
The reported IC50 values represent the means ± standard deviations (n = 2).
2.9. NBD compounds are HIV-1 entry-antagonist
The HIV-1 inhibitor NBD-556[7] was afterward described as an entry agonist because it promotes CCR5 binding, enhancing HIV-1 entry into CD4-negative cells expressing CCR5[9, 10]. To verify if the new five inhibitors behave as an entry antagonist, we infected CD4-negative Cf2Th-CCR5 cells with the recombinant CD4-dependant HIV-1ADA virus in the presence of escalating concentrations of the compounds. NBD-556 (a proven HIV-1 entry agonist) and NBD-14270 (a proven HIV-1 entry antagonist[11]) were used as a control. Results are reported in Figure 7 and expressed as relative virus infectivity, which designates the ratio of the amount of infection detected in the presence of the compounds and the amount of infection detected in the absence of the compounds. As expected, NBD-556 enhanced the infection of the Cf2Th-CCR5 cells in a dose-dependent manner, while the control NBD-14270 and the five compounds did not enhance HIV-1 infectivity in these cells, indicating that the HIV-1 entry antagonist property is maintained. In Figure 7, data is shown for NBD-556, NBD-14270, 12(fR), 13(fR); data for 12(fS), 13(fS), and 17 is not shown.
Figure 7.
Infectivity of Cf2Th−CCR5 cells by CD4-dependent HIV-1ADA. Cf2Th−CCR5 cells were infected with CD4-dependent HIV-1ADA in the presence of 12(fR) and 13(fR). NBD-556 and NBD-14270 were used as a control. The Relative virus infectivity designates the ratio of the amount of infection detected in the presence of the compounds and the amount of infection detected in the absence of the compounds. Three independent experiments were performed in triplicate, and the graph is representative of one experiment. The toxicity of the compounds against these cells was evaluated to calculate the CC50 values: for NBD-556, the CC50 was > 60, for NBD-14270 > 47, for 12(fR) >86 and for 13(fR) 24.7±1.8. All the values represent the mean ± standard deviation.
3.0. Experimental
3.0.1. Reagents and materials
Reagents were purchased at the highest commercial quality and used without further purification unless otherwise stated. Starting materials 1b, 5a-6a, 7a-b, 8a-b, 9b, 10b, 11b-12b, 13a-17a and 18a kindly provided by EDASA scientific commercial building block reagents. Reactions were monitored by thin-layer chromatography (TLC) carried out on Merck TLC Silica gel plates (60F254), using a UV light for visualization and basic aqueous potassium permanganate or iodine fumes as developing agents. 1H and 13C NMR spectra were recorded on Bruker Avance 400 instrument with an operating frequency of 400 and 100 MHz respectively and calibrated using residual undeuterated chloroform (δH = 7.26 ppm) and CDCl3 (δC = 77.16 ppm), or undeuterated DMSO (δH = 2.50 ppm) and DMSO-d6 (δC = 39.51 ppm) as internal references. The following abbreviations are used to set multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet, br. = broad. IR spectra were recorded on Thermo Nicolet IR-200 in KBr, nujol or neat. High-resolution mass spectra (HRMS) were recorded on the Thermo Scientific LTQ Orbitrap instrument using nanoelectrospray ionization (nano-ESI). Low-resolution mass spectra were on Finnigan MAT mass spectrometer using electron ionization (direct inlet) and an ITD-700 detector with the ionizing electron energy being 70 eV and the mass range being m/z 35–400. Elemental analysis was performed on EURO EA CHN Elemental Analyzer. The melting points (m.p.) were measured in open capillaries and presented without correction.
3.0.2. GLIDE-based docking
We used the automated docking software GLIDE 8.9 within maestro 12.6 (Schrödinger, Portland, OR) in Schrödinger Suite 2020–4[25, 26] that uses a hierarchical series of filters in searching for appropriate ligand conformation in the active site of a target protein. It uses a funnel-shaped scoring process to sort out the best conformations and orientations of the ligand (defined as pose) based on its interactions with the receptor. GLIDE has been used successfully in drug design[27–31].
We used the crystal structure of clade A/E HIV-1 gp120 core in complex with NBD-14010 (PDB: 5U6E)[2] that we reported earlier as the target protein. We used the “Protein Preparation Wizzard” within maestro in the Schrodinger suite 2020–4 to optimize hydrogens, bond orders, charges, and steric clashes using the OPLS3e force field[32]. We used this optimized protein structure to create a grid file encompassing the area in the cavity using NBD-14010 as the ligand.
We generated three-dimensional coordinates of the ligands, their isomeric, ionization, and tautomeric states using the LigPrep (including Ionizer) module within the Schrödinger Suite 2020–4. Conformational flexibility of the ligands was handled via an exhaustive conformational search. Initially, we used Schrödinger’s proprietary GlideScore scoring function in extra precision (XP) mode to score the optimized poses.
3.1. Chemistry
3.1.1. General Procedure A: for Amide Coupling
DIPEA (1 equiv.) was added to an appropriate acid (1 equiv.) followed by dissolving in DMF (10 mL per 1 g of acid) and then HBTU (1 equiv.). The resulting solution was stirred for 1–3 min and added to a solution of appropriate amine (1 equiv.) in DMF (10 mL per 1 g of amine) in several portions. The reaction mixture was stirred overnight; DMF was evaporated, and the residue was dissolved in CH2Cl2 (50 mL per 1 g of crude product) and successively washed with 5% aqueous NaOH and 10% tartaric acid solutions (25 mL per 1 g of crude product). The organic layer was dried over anhydrous Na2SO4, filtered, evaporated, and dry loaded on silica. The crude product was purified by flash column chromatography on silica gel (eluent: hexane-EtOAc, 3:1 →1:1 → 0:1). Products were used in the next step without analysis.
3.1.2. General Procedure B: for Allyl- and Alloc- Deprotection
To a solution containing protected compound (1 equiv.) and N,N-dimethyl barbituric acid (NDMBA, 3 equiv.) in MeOH (0.1M solution), PPh3 (10 mol %) was added under a nitrogen atmosphere followed by Pd(dba)2 (5 mol %). The mixture was stirred for 1 day under reflux. After cooling, 50 mL of CH2Cl2 was added, and the organic phase was shaken with 10% aqueous K2CO3 (50 mL) to remove the unreacted NDMBA. The organic layer was separated, and the aqueous layer was extracted with CH2Cl2-EtOH (∼4:1, (4−5) × 50 mL). Combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. Purification by flash chromatography (twice) (eluent 1: CHCl3-MeOH (saturated with NH3∼7M), 10:1 and 5:1, eluent 2: CH2Cl2-MeOH, 4:1, 2:1, 1:1) afforded amine as a slightly yellow or white solid.
3.1.3. N1-(2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)ethyl)-N2-(4-chlorophenyl)oxalamide (1)
Compounds 1(fR), NBD-14081, and 1(fS), NBD-14082 were obtained following the general procedure A and B from amines 1a(fR), 1a(fS), and acid 1b. Both compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1).
1(fR), NBD-14081: M = 156 mg. Yield = 24% (over two steps). rt = 1.164 min. Purity = 100%. LC-MS: m/z [M+H]+ = 369 Da.
1(fS), NBD-14082: M = 291 mg. Yield = 39% (over two steps). rt = 1.174 min. Purity = 99%. LC-MS: m/z [M+H]+ = 369 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 1.75 (br. s., 2 H), 2.25 (s, 3 H), 3.06 (d, J=6.36 Hz, 2 H), 4.54 (s, 2 H), 5.01 (t, J=5.72 Hz, 1 H), 5.39 (br. s., 1 H), 7.42 (d, J=8.90 Hz, 2 H), 7.87 (d, J=8.90 Hz, 2 H), 9.41 (br. s., 1 H), 10.84 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 14.9, 45.1, 54.9, 55.1, 122.1 (2C), 128.4, 128.7 (2C), 132.9, 136.6, 146.9, 158.4, 160.2, 167.2.
HRMS (ESI): m/z calcd for C15H18ClN4O3S [M+H]+ 369.0783, found 369.0793.
3.1.4. N1-(3-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)propyl)-N2-(4-chlorophenyl)oxalamide (2)
Compounds 2(fR), NBD-14079 and 2(fS), NBD-14080 were obtained following the general procedure A and B from amines 2a(fR), 2a(fS), and acid 1b. Compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1).
2(fR), NBD-14079: M = 323 mg. Yield = 22% (over two steps). rt = 1.138 min. Purity = 100%. LC-MS: m/z [M+H]+ = 383 Da.
2(fS), NBD-14080: M = 177 mg. Yield = 23% (over two steps). rt = 1.071 min. Purity = 100%. LC-MS: m/z [M+H]+ = 383 Da.
1 H NMR (DMSO-d6, 400 MHz): δ= 2.05 (q, J=6.4 Hz, 2 H), 2.26 (s, 3 H), 2.53 – 2.63 (m, 1 H), 2.65 – 2.73 (m, 1 H), 4.55 (s, 2 H), 5.25 (t, J=6.8 Hz, 1 H), 5.42 (br. s., 1 H), 7.43 (d, J=8.9 Hz, 2 H), 7.87 (d, J=8.9 Hz, 2 H). (Four exchangeable protons are missing).
13C NMR (DMSO-d6, 100 MHz): δ= 14.8, 36.6, 38.1, 50.3, 55.0, 122.0 (2C), 128.3, 128.6 (2C), 132.7, 136.6, 146.8, 158.4, 159.9, 168.8.
HRMS (ESI): m/z calcd for C16H20ClN4O3S [M+H]+ 383.0939, found 383.0939.
3.1.5. Ethyl 2-[(4-chloro-3-fluoro-phenyl)methylamino]-2-oxo-acetate (3b)
Amine salt (16.68 g. 85.1 mmol) was suspended in CH2Cl2 (170 mL), and Et3N (30 mL, 215 mmol, 2.5 equiv) was added. Ethyl chlorooxoacetate (11.4 mL, 102 mmol, 1.2 equiv) was added dropwise. The reaction mixture was stirred for 1 h and diluted with 5% aqueous HCl (200 mL). The organic layer was separated, dried over Na2SO4, filtered, and evaporated. The residue was used without further purification. If necessary, the product can be purified by column chromatography (eluent: 3:1, 1:1 hexanes\EtOAc). M=10.17 g. Yield=46%.
1H NMR: (CDCl3, 400 MHz) δ = 1.41 (t, J=7.2 Hz, 3 H), 4.37 (q, J=7.0 Hz, 2 H), 4.51 (d, J=6.4 Hz, 2 H), 7.05 (d, J=8.3 Hz, 1 H), 7.12 (dd, J=9.7, 1.4 Hz, 1 H), 7.38 (t, J=7.8 Hz, 1 H), 7.55 (br. s, 1 H).
13C NMR: (CDCl3, 100 MHz) δ = 14.0, 42.9, 63.5, 116.2 (d, J=21.7 Hz), 120.4 (d, J=17.7 Hz), 124.3 (d, J=3.2 Hz), 130.9, 137.9 (d, J=6.4 Hz), 156.8, 158.1 (d, J=249.8 Hz), 160.5.
3.1.6. 2-[(4-chloro-3-fluoro-phenyl)methylamino]-2-oxo-acetic acid (3c)
Appropriate ethyl ester 3b (10.17 g, 39.1 mmol, 1 equiv.) was added to a solution of NaOH (3.14 g, 78.5 mmol, 2 equiv.) in EtOH-H2O mixture (1:1, 80 mL). The resulting reaction mixture was refluxed for 5–6 h (TLC-control) and cooled to room temperature. A concentrated aqueous HCl solution (~12 M, 6.5 mL, 78.5 mmol, 2 equiv.). The reaction mixture was diluted with water (100 mL), the formed precipitate was filtered and washed with water (2 × 50 mL). The pure product was dried under reduced pressure. M = 4.21 g. Yield = 46%.
1H NMR (DMSO-d6, 400 MHz): δ= 4.30 (d, J=6.4 Hz, 2 H), 7.12 (d, J=8.2 Hz, 1 H), 7.28 (dd, J=10.4, 1.3 Hz, 1 H), 7.51 (t, J=8.0 Hz, 1 H), 8.61 (br. s., 1 H), 9.32 (t, J=6.1 Hz, 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 41.6, 115.7 (d, J=20.9 Hz), 117.8 (d, J=17.7 Hz), 124.6 (d, J=3.2 Hz), 130.5, 140.8 (d, J=6.4 Hz), 157.1 (d, J=246.6 Hz), 159.8, 162.3.
3.1.7. N’-[2-amino-1-[5-(hydroxymethyl)thiazol-2-yl]ethyl]-N-[(4-chloro-3-fluorophenyl) methyl]oxamide (3)
Compounds 3(fR), NBD-14314 and 3(fS), NBD-14313 were obtained following the general procedure A and B from amines 1a(fR) and 1a(fS), and acid 3c. Compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1).
3(fR), NBD-14314: M = 200 mg. Yield = 21% (over two steps). rt = 1.214 min. Purity = 100%. LC-MS: m/z [M+H]+ = 387 Da.
3(fS), NBD-14313: M = 195 mg. Yield = 19% (over two steps). rt = 1.198 min. Purity = 100%. LC-MS: m/z [M+H]+ = 387 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 1.74 (br. s., 2 H), 3.00 – 3.08 (m, 2 H), 4.28 – 4.41 (m, 2 H), 4.61 (d, J=5.5 Hz, 2 H), 4.98 – 5.04 (m, 1 H), 5.49 (t, J=5.7 Hz, 1 H), 7.15 (dd, J=8.2, 1.3 Hz, 1 H), 7.32 (dd, J=10.3, 1.8 Hz, 1 H), 7.51 – 7.58 (m, 2 H), 9.28 (br. s., 1 H), 9.48 (t, J=6.5 Hz, 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 41.6, 45.1, 54.7, 55.7, 115.8 (d, J=20.9 Hz), 117.9 (d, J=17.7 Hz), 124.6 (d, J=3.2 Hz), 130.5, 139.0, 140.3, 140.5 (d, J=6.4 Hz), 157.0 (d, J=246.6 Hz), 160.0, 160.2, 170.0.
HRMS (ESI) calcd for C15H17ClF6N4O3S [M +H]+ 387.0688, found 387.0690.
3.1.8. N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-2-((4-chlorobenzyl)amino)acetamide dihydrochloride (4)
Compounds 4(fR), NBD-14155 and 4(fS), NBD-14154 were obtained following the general procedure A and B and followed by Boc-deprotection (see paragraph below) from amines 3a(fR), 3a(fS), and acid 4b. The resulting final compounds were purified after allyl- and alloc-deprotection (gen. proc. B) using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1).
Boc-deprotection:
To Boc-protected compound was added 50 mL of 1 M HCl in methanol. The solution was stirred for 2 h at room temperature, and then the solvent was evaporated. The precipitate was triturated in Et2O (50 mL) and filtered. The product was washed with Et2O (2 × 50 mL) and CH2Cl2 (2 × 50 mL) and dried on a rotary evaporator under reduced pressure.
4(fR), NBD-14155: M = 481 mg. Yield = 41% (over two steps). rt = 0.663 min. Purity = 97%. LC-MS: m/z [M+H]+ = 355 Da.
4(fS), NBD-14154: M = 552 mg. Yield = 55% (over two steps). rt = 0.793 min. Purity = 99%. LC-MS: m/z [M+H]+ = 355 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 3.19 – 3.30 (m, 1 H), 3.38 – 3.51 (m, 1 H), 3.70 – 3.88 (m, 2 H), 4.13 – 4.25 (m, 2 H), 4.63 (s, 2 H), 5.42 – 5.50 (m, 1 H), 7.49 (d, J=8.31 Hz, 2 H), 7.55 – 7.63 (m, 3 H), 8.57 (br. s., 3 H), 9.69 (d, J=8.19 Hz, 1 H), 9.81 (br. s., 2 H). (One exchangeable proton is missing)
13C NMR (DMSO-d6, 100 MHz): δ= 41.4, 47.0, 49.0, 49.1, 55.7, 128.7 (2 C), 130.6, 132.4 (2 C), 133.9, 139.1, 142.0, 165.7, 167.2.
HRMS (ESI): m/z calcd for C15H19ClN4O2S [M+H]+ 355.0990, found 355.0993.
3.1.9. 4-Chlorobenzamide (5b)
4-Chlorobenzoic acid (14.8 g, 94.9 mmol, 1 equiv.) was dissolved in CHCl3 (95 mL), Et3N (15.54 mL, 104.6 mmol, 1.1 equiv.) was added in a single portion followed by dropwise addition of ethyl chloroformate (10.0 mL, 104.6 mmol, 1.1 equiv.). After the addition, the mixture was cooled to 0 °C, and aqueous ammonia (50 mL) was slowly added. The mixture was stirred for 1 h at RT, and layers were separated. The aqueous phase was extracted with CH2Cl2 (2 × 50 mL). Combined organic layers were washed with water (50 mL), brine (50 mL), dried over anhydrous Na2SO4, filtered, and evaporated. The product was used in the next step without further purification. M = 7.90 g. Yield = 54%.
1H NMR: (DMSO-d6, 400 MHz) δ = 7.47 (br. s, 1 H), 7.52 (d, J=8.3 Hz, 2 H), 7.89 (d, J=8.3 Hz, 2 H), 8.06 (br. s, 1 H).
3.1.10. 4-Chlorobenzoyl isocyanate (5c)
Amide 24 (7.9 g, 51.0 mmol, 1 equiv.) was dissolved in 1,2-dichloroethane (51 mL), and oxalyl chloride (22 mL, 260 mmol, 5.0 equiv) was added in a single portion. The mixture was heated at reflux for 1 day and cooled to room temperature. The resulting solution was evaporated to dryness. The product was used in the next step without further purification. M = 9.23 g. Yield = 100%.
1H NMR (CDCl3, 400 MHz): δ = 7.47 (d, 2 H), 8.01 (d, J=7.9 Hz, 2 H).
13C NMR (DMSO-d6, 100 MHz): δ = 129.3 (2 C), 129.9, 131.6, 131.9 (2 C), 144.6, 164.3.
3.1.11. N-((2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)ethyl)carbamoyl)-4-chlorobenzamide (5)
To a solution of amine 1a (0.7 g, 2.25 mmol, 1 equiv.) in MeCN (10 mL), 4-chlorobenzoyl isocyanate 5c (0.45 g, 2.48 mmol, 1.1 equiv.) was added, followed by a catalytic amount of DMAP. The mixture was stirred for 30 min at room temperature and then refluxed for 4–5 h (TLC-control). After that, the solvent was evaporated, and the residue was dissolved in CH2Cl2 (50 mL) and washed with 10% aqueous tartaric acid (25 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, evaporated. The crude product was purified by flash column chromatography on silica gel (eluent: hexane-EtOAc, 3:1 →1.1 → 0:1). The product was used without analysis.
Allyl- and alloc-deprotection was carried out according to the general procedure B.
5(fR), (NBD-14087): M = 117 mg. Yield = 15% (over two steps). rt = 1.134 min. Purity = 95%. LC-MS: m/z [M+H]+ = 369 Da.
5(fS), (NBD-14087): M = 245 mg. Yield = 29% (over two steps). rt = 1.076 min. Purity = 92%. LC-MS: m/z [M+H]+ = 369 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 2.27 (s, 3 H), 2.95 – 3.02 (m, 1 H), 3.06 – 3.13 (m, 1 H), 3.35 (br. s, 3H), 4.55 (s, 2 H), 4.98 – 5.07 (m, 1 H), 5.41 (br. s., 1 H), 7.59 (d, J=8.58 Hz, 2 H), 8.00 (d, J=8.58 Hz, 2 H), 9.40 (d, J=7.95 Hz, 1 H). (Three exchangeable proton signals are missing)
13C NMR (DMSO-d6, 100 MHz): δ= 14.9, 45.5, 53.8, 55.1, 128.7 (2 C), 130.2 (2 C), 131.3, 132.9, 137.8, 147.1, 153.3, 167.3, 168.1.
HRMS (ESI): m/z calcd for C15H18ClN4O3S [M+H]+ 369.0783, found 369.0782.
3.1.12. 1-(5-cyclohexyl-1H-pyrrol-2-yl)-2,2,2-trifluoroethanone (6b)
Pyrrole 6a (8.95 g, 60.0 mmol, 1 equiv.) was dissolved in CH2Cl2 (120 mL), and pyridine (5.83 mL, 72.1 mmol, 1.2 equiv.) was added, followed by dropwise addition of TFAA (10.02 mL, 72.1 mmol, 1.2 equiv.). After completion of the addition, the mixture was stirred for 2–3 h at room temperature, and the solvent was evaporated. The crude product was purified by flash chromatography on silica gel (eluent: hexane-EtOAc, 30:1 → 20:1). M = 9.58 g. Yield = 65%.
1H NMR: (CDCl3, 400 MHz) δ = 1.20 – 1.33 (m, 1H), 1.33 – 1.54 (m, 4H), 1.69 – 1.79 (m, 1H), 1.80 – 1.89 (m, 2H), 1.96 – 2.09 (m, 2H), 2.75 (tt, J=11.3, 3.2 Hz, 1H), 6.18 (dd, J=4.0, 2.6 Hz, 1H), 7.12 – 7.25 (m, 1H), 10.49 (br. s, 1H).
13C NMR: (CDCl3, 100 MHz) δ = 25.8, 26.1, 32.5, 37.5, 109.8, 117.5 (q, J=288.3 Hz), 123.7 (q, J=3.4 Hz), 124.9, 152.6, 168.9, 169.1 (q, J=35.9 Hz).
3.1.13. 5-cyclohexyl-1H-pyrrole-2-carboxylic acid (6c)
Appropriate trifluoroethanone 6b (2.58 g, 10.5 mmol, 1 equiv.) was added to a solution of NaOH (2.1 g, 52.6 mmol, 5 equiv.) in EtOH-H2O mixture (1:1, 53 mL). The resulting reaction mixture was refluxed for 20 h and cooled to room temperature. A concentrated aqueous HCl solution (~12 M, 5 equiv.) was added dropwise, and water (50 mL) was added. The resulting mixture was extracted with CH2Cl2 (3 × 100 mL). Combined organic phases were dried over anhydrous Na2SO4, filtered, and evaporated. The crude product was purified by flash chromatography on silica gel (eluent: hexane-EtOAc, 1:1 → 0:1) to afford 5. M = 1.69 g. Yield = 83%.
1H NMR (CDCl3, 400 MHz): δ= 1.22 – 1.48 (m, 5 H), 1.68 – 1.87 (m, 3 H), 1.94 – 2.05 (m, 2 H), 2.55 – 2.70 (m, 1 H), 5.99 – 6.07 (m, 1 H), 6.96 – 7.03 (m, 1 H), 9.14 (br. s., 1 H), 10.92 (br. s., 1 H).
13C NMR (CDCl3, 400 MHz): δ= 26.0, 26.2 (2 C), 32.9 (2 C), 37.1, 107.1, 118.3, 119.9, 145.6, 166.1.
3.1.14. N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-5-cyclohexyl-1H-pyrrole-2-carboxamide (6)
Compounds 6(fR), NBD-14146 and 6(fS), NBD-14147 were respectively obtained following the general procedure A and B from amines 3a(fR), 3a(fS), and acid 6c. Compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1).
6(fR), NBD-14146: M = 87 mg. Yield = 8% (over two steps). rt = 1.222 min. Purity = 95%. LC-MS: m/z [M+H]+ = 349 Da.
6(fS), NBD-14147: M = 275 mg. Yield = 16% (over two steps). rt = 1.055 min. Purity = 96%. LC-MS: m/z [M+H]+ = 349 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 1.11 – 1.42 (m, 5 H), 1.45 – 1.92 (m, 7 H), 2.53 – 2.61 (m, 1 H), 2.97 (dd, J=13.14, 7.89 Hz, 1 H), 3.10 (dd, J=13.20, 5.26 Hz, 1 H), 4.60 (s, 2 H), 5.10 – 5.19 (m, 1 H), 5.47 (br. s., 1 H), 5.81 – 5.85 (m, 1 H), 6.77 – 6.82 (m, 1 H), 7.53 (s, 1 H), 8.29 (d, J=7.95 Hz, 1 H), 11.17 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 25.6, 25.9 (2 C), 32.7, 32.7, 36.3, 45.7, 54.3, 55.8, 104.1, 111.3, 124.1, 139.0, 139.9, 142.4, 160.8, 172.5.
HRMS (ESI): m/z calcd for C21H30N3O3S [M+H]+ 349.1693, found 349.1695.
3.1.15. Methyl 4-(cyclohexylmethoxy)benzoate (7c)
Methyl 4-hydroxybenzoate 7a (19.78 g, 130 mmol, 1 equiv.) and cyclohexylmethyl methanesulfonate 7b (25 g, 130 mmol, 1 equiv.) were dissolved in DMF (260 mL). Solid K2CO3 (35.89 g, 260 mmol, 2 equiv.) was added to a solution followed by NaI*2H2O (2.42 g, 13.0 mmol, 0.1 equiv.) and TBAB (4.19 g, 13.0 mmol, 0.1 equiv.) addition. The reaction mixture was stirred for 12–15 h at 90–100 °C on an oil bath. After cooling to room temperature, the mixture was poured into water (550 mL) and extracted with EtOAc (3 × 200 mL). Combined organic phases were washed with water (150 mL) and brine (2 × 100 mL), dried over anhydrous Na2SO4, filtered, and evaporated. The crude product was purified by flash chromatography on silica gel (eluent: hexane-EtOAc, 20:1 → 10:1). M = 24.57 g. Yield = 76%.
1H NMR (CDCl3, 400 MHz): δ= 0.99 – 1.13 (m, 2 H), 1.17 – 1.38 (m, 3 H), 1.65 – 1.93 (m, 6 H), 3.80 (d, J=6.24 Hz, 2 H), 3.88 (s, 3 H), 6.90 (d, J=8.56 Hz, 2 H), 7.98 (d, J=8.56 Hz, 2 H).
13C NMR (CDCl3, 100 MHz): δ= 25.8 (2 C), 26.5, 29.9 (2 C), 37.7, 51.8, 73.6, 114.1 (2 C), 122.3, 131.6 (2 C), 163.2, 167.0.
3.1.16. Methyl 4-((cyclohexylmethyl)amino)benzoate (8c)
To a solution of methyl 4-aminobenzoate (34.7 g, 230 mmol, 1 equiv.) in CH2Cl2 (460 mL) on an ice bath, cyclohexanecarbaldehyde (25.75 g, 230 mmol, 1 equiv.) was added, followed by dropwise addition of AcOH (1.31 mL, 23.0 mmol, 0.1 equiv.). The mixture was stirred for 10–15 min, and then NaBH(OAc)3 (121.6 g, 574 mmol, 2.5 equiv.) was added portion-wise at the same temperature. The resulting mixture was stirred for 1 h at 0–5 °C and 10–12 h at room temperature; after that, it was poured (carefully! CO2 evolution) into 10% aqueous K2CO3 (1000 mL). The organic layer was separated, and water was extracted with CH2Cl2 (3 × 150 mL). Combined organic phases were washed with brine (200 mL), dried over anhydrous Na2SO4, filtered, and evaporated. The crude product was purified by flash column chromatography on silica gel (eluent: hexane-EtOAc, 20:1 → 10:1). M = 45.29 g. Yield = 80%.
1H NMR (CDCl3, 400 MHz): δ= 0.91 – 1.03 (m, 2 H), 1.12 – 1.31 (m, 3 H), 1.51 – 1.84 (m, 6 H), 2.99 (d, J=6.72 Hz, 2 H), 3.84 (s, 3 H), 4.28 (br. s., 1 H), 6.50 – 6.55 (m, 2 H), 7.82 – 7.87 (m, 2 H).
13C NMR (CDCl3, 100 MHz): δ= 26.0 (2 C), 26.5, 31.2 (2 C), 37.6, 49.9, 51.5, 111.3 (2 C), 117.7, 131.6 (2 C), 152.4, 167.5.
3.1.17. 4-(cyclohexylmethoxy)benzoic acid (7d)
Appropriate methyl ester 7c (24.57 g, 98.9 mmol, 1 equiv.) was added to a solution of NaOH (7.92 g, 198 mmol, 2 equiv.) in EtOH-H2O mixture (1:1, 200 mL). The resulting reaction mixture was refluxed for 7–8 h (TLC-control) and cooled to room temperature. A concentrated aqueous HCl solution (~12 M, 16.5 mL, 2 equiv.) was added dropwise, and water (200 mL) was added. The precipitate that formed was filtered, washed with water (2 × 50 mL). The crude product was recrystallized from EtOH. M = 21.39 g. Yield = 92%.
1H NMR (DMSO-d6, 400 MHz): δ= 0.90 – 1.06 (m, 2 H), 1.09 – 1.31 (m, 3 H), 1.55 – 1.83 (m, 6 H), 3.78 (d, J=6.11 Hz, 2 H), 6.96 (d, J=8.80 Hz, 2 H), 7.88 (d, J=8.68 Hz, 2 H), 12.60 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 25.3 (2 C), 26.1, 29.2 (2 C), 37.0, 73.0, 114.2 (2 C), 122.8, 131.4 (2 C), 162.5, 167.1.
3.1.18. 4-((cyclohexylmethyl)amino)benzoic acid (8d)
Appropriate methyl ester 8c (5.76 g, 23.3 mmol, 1 equiv.) was added to a solution of NaOH (1.86 g, 46.6 mmol, 2 equiv.) in EtOH-H2O mixture (1:1, 35 mL). The resulting reaction mixture was refluxed for 5–6 h (TLC-control) and cooled to room temperature. A concentrated aqueous HCl solution (~12 M, 3.88 mL, 2 equiv.) was added dropwise, and water (50 mL) was added. The precipitate that formed was filtered, washed with water (2 × 50 mL). The pure product was dried under reduced pressure. M = 4.71 g. Yield = 87%.
1H NMR (DMSO-d6, 400 MHz): δ= 0.85 – 0.98 (m, 2 H), 1.08 – 1.24 (m, 3 H), 1.44 – 1.82 (m, 6 H), 2.89 (t, J=6.05 Hz, 2 H), 6.44 (t, J=5.44 Hz, 1 H), 6.54 (d, J=8.68 Hz, 2 H), 7.64 (d, J=8.68 Hz, 2 H), 11.88 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 25.6 (2 C), 26.2, 30.7 (2 C), 36.9, 49.0, 110.7 (2 C), 116.4, 131.3 (2 C), 153.0, 167.7.
3.1.19. N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-4-(cyclohexylmethoxy)benzamide (7)
Compounds 7(fR), NBD-14216 and 7(fS), NBD-14217) were obtained following the general procedure A and B from amines 3a(fR), 3a(fS), and acid 7d. Compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1).
7(fR), NBD-14216: M = 596 mg. Yield = 53% (over two steps) rt = 1.357 min. Purity = 100%. LC-MS: m/z [M+H]+ = 390 Da.
7(fS), NBD-14217): M = 610 mg. Yield = 47% (over two steps). rt = 1.363 min. Purity = 100%. LC-MS: m/z [M+H]+ = 390 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 0.97 – 1.10 (m, 2 H), 1.13 – 1.32 (m, 3 H), 1.49 – 1.92 (m, 8 H), 3.00 (dd, J=13.08, 8.07 Hz, 1 H), 3.11 (dd, J=13.20, 5.01 Hz, 1 H), 3.85 (d, J=5.99 Hz, 2 H), 4.60 (s, 2 H), 5.08 – 5.24 (m, 1 H), 5.47 (br. s., 1 H), 7.01 (d, J=8.56 Hz, 2 H), 7.54 (s, 1 H), 7.89 (d, J=8.68 Hz, 2 H), 8.79 (d, J=7.21 Hz, 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 25.3 (2 C), 26.0, 29.2 (2 C), 37.0, 45.6, 55.3, 55.8, 72.9, 113.9 (2 C), 126.1, 129.4 (2 C), 139.0, 139.9, 161.4, 166.2, 172.2.
HRMS (ESI): m/z calcd for C20H28N3O3S [M+H]+ 390.1846, found 390.1850.
3.1.20. N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-4-((cyclohexylmethyl)amino)benzamide (8)
Compounds 8(fR), NBD-14218 and 8(fS), NBD-14219 were obtained following the general procedure A and B from amines 3a(fR), 3a(fS), and acid 8d. Compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1).
8(fR), NBD-14218: M = 785 mg. Yield = 44% (over two steps). rt = 1.366 min. Purity = 100%. LC-MS: m/z [M+H]+ = 389 Da.
8(fS), NBD-14219: M = 684 mg. Yield = 38% (over two steps). rt = 1.338 min. Purity = 98%. LC-MS: m/z [M+H]+ = 389 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 0.87 – 1.01 (m, 2 H), 1.10 – 1.26 (m, 3 H), 1.39 – 1.86 (m, 8 H), 2.91 (t, J=6.19 Hz, 2 H), 3.00 (dd, J=13.14, 7.83 Hz, 1 H), 3.10 (dd, J=13.14, 5.05 Hz, 1 H), 4.60 (s, 2 H), 5.16 (td, J=7.64, 5.31 Hz, 1 H), 5.47 (br. s., 1 H), 6.25 (t, J=5.62 Hz, 1 H), 6.58 (d, J=8.72 Hz, 2 H), 7.53 (s, 1 H), 7.70 (d, J=8.72 Hz, 2 H), 8.46 (d, J=7.71 Hz, 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 25.6 (2 C), 26.2, 30.7 (2 C), 36.9, 45.7, 49.0, 55.0, 55.8, 110.5 (2 C), 120.1, 129.1 (2 C), 139.0, 139.8, 151.9, 166.7, 172.9.
HRMS (ESI): m/z calcd for C20H29N4O2S [M+H]+ 389.2006, found 389.2009.
3.1.21. N-(2-amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-2-(4-(cyclohexylmethoxy)phenyl)acetamide (9)
Compounds 9(fR), NBD-14157 and 9(fS), NBD-14156 were obtained following the general procedure A and B from amines 3a(fR), 3a(fS), and acid 9b. Compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1). 9(fR), NBD-14157: M = 35 mg. Yield = 8% (over two steps). rt = 1.443 min. Purity = 93%. LC-MS: m/z [M+H]+ = 404 Da.
9(fS), NBD-14156: M = 448 mg. Yield = 50% (over two steps). rt = 1.402 min. Purity = 94%. LC-MS: m/z [M+H]+ = 404 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 0.95 – 1.08 (m, 2 H), 1.12 – 1.31 (m, 3 H), 1.53 – 1.88 (m, 8 H), 2.86 (dd, J=13.08, 7.34 Hz, 1 H), 2.98 (dd, J=13.14, 5.20 Hz, 1 H), 3.44 (s, 2 H), 3.73 (d, J=6.36 Hz, 2 H), 4.59 (d, J=5.26 Hz, 2 H), 4.87 – 4.99 (m, 1 H), 5.48 (t, J=5.56 Hz, 1 H), 6.84 (d, J=8.44 Hz, 2 H), 7.17 (d, J=8.44 Hz, 2 H), 7.52 (s, 1 H), 8.64 (d, J=7.82 Hz, 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 25.3 (2 C), 26.1, 29.3 (2 C), 37.1, 41.3, 45.9, 54.4, 55.7, 72.7, 114.2, 127.9, 130.0, 139.1, 139.9, 157.5, 170.7, 171.6.
HRMS (ESI): m/z calcd for C21H30N3O3S [M+H]+ 404.2002, found 404.2002.
3.1.22. N-(2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)ethyl)-1H-indole-2-carboxamide (10)
Compounds 10(fR), NBD-14032 and 10(fS), NBD-14033 were obtained following the general procedure A and B from amine 1a(fR), 1a(fS), and acid 10b. Compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1).
10(fR), NBD-14032: M = 58 mg. Yield = 6% (over two steps). rt = 1.000 min. Purity = 100%. LC-MS: m/z [M+H]+ = 331 Da.
10(fS), NBD-14033: M = 534 mg. Yield = 32% (over two steps). rt = 1.170 min. Purity = 99%. LC-MS: m/z [M+H]+ = 331 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 2.25 (s, 3 H), 3.02 (dd, J=13.3, 8.1 Hz, 1 H), 3.13 (dd, J=13.1, 5.2 Hz, 1 H), 3.33 (br. s., 2 H), 4.52 (s, 2 H), 5.10 – 5.23 (m, 1 H), 5.37 (br. s., 1 H), 7.04 (t, J=7.5 Hz, 1 H), 7.19 (t, J=7.6 Hz, 1 H), 7.29 (s, 1 H), 7.43 (d, J=8.2 Hz, 1 H), 7.63 (d, J=7.9 Hz, 1 H), 8.91 (d, J=6.5 Hz, 1 H), 11.62 (s, 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 14.9, 45.5, 54.5, 55.0, 103.5, 112.3, 119.8, 121.6, 123.5, 127.0, 131.2, 132.6, 136.6, 146.9, 161.4, 169.0.
HRMS (ESI): m/z calcd for C16H19N4O2S [M+H]+ 331.1223, found 331.1221.
3.1.23. N-(2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)ethyl)-5-chloro-1H-indole-2-carboxamide (11)
Compounds 11(fR), NBD-14030 and 11(fS), NBD-14031 were obtained following the general procedure A and B from amine 1a(fR), 1a(fS), and acid 11b. Compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1).
11(fR), NBD-14030: M = 91 mg. Yield = 10% (over two steps). rt = 1.137 min. Purity = 98%. LC-MS: m/z [M+H]+ = 365 Da.
11(fS), NBD-14031: M = 616 mg. Yield = 34% (over two steps). rt = 1.327 min. Purity = 100%. LC-MS: m/z [M+H]+ = 365 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 2.25 (s, 3 H), 3.03 (dd, J=13.2, 8.3 Hz 1 H), 3.15 (dd, J=13.1, 5.1 Hz, 1 H), 3.17 (br. s, 2H), 4.52 (s, 2 H), 5.16 – 5.25 (m, 1 H), 5.37 (br. s., 1 H), 7.19 (dd, J=8.8, 2.0 Hz, 1 H), 7.28 (s, 1 H), 7.43 (d, J=8.8 Hz, 1 H), 7.73 (d, J=1.8 Hz, 1 H), 9.03 (s, 1 H), 11.84 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 14.9, 45.2, 54.2, 55.0, 103.1, 113.9, 120.7, 123.7, 124.3, 128.1, 132.6, 132.7, 135.0, 147.0, 161.0, 168.6.
HRMS (ESI): m/z calcd for C16H18ClN4O2S [M+H]+ 365.0834, found 365.0830.
3.1.24. N-(2-amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-6-chloro-1H-indole-2-carboxamide (12)
Compounds 12(fR), NBD-14294 and 12(fS), NBD-14293 were obtained following the general procedure A and B from amine 12a(fR), 12a(fS), and acid 12b. Compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1).
12(fR), NBD-14294: M = 516 mg. Yield = 35% (over two steps). rt = 1.109 min. Purity = 100%. LC-MS: m/z [M+H]+ = 351 Da.
12(fS), NBD-14293: M = 423 mg. Yield = 36% (over two steps). rt = 1.109 min. Purity = 100%. LC-MS: m/z [M+H]+ = 351 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 1.72 (br. s., 2 H), 3.02 (dd, J=13.25, 7.99 Hz, 1 H), 3.15 (dd, J=13.32, 5.33 Hz, 1 H), 4.54 (d, J=4.80 Hz, 2 H), 5.24 (br. s., 1 H), 5.28 – 5.34 (m, 1 H), 7.07 (dd, J=8.53, 1.90 Hz, 1 H), 7.29 (s, 1 H), 7.35 (s, 1 H), 7.44 (d, J=1.37 Hz, 1 H), 7.69 (d, J=8.53 Hz, 1 H), 9.03 (br. s., 1 H), 11.80 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 45.7, 54.8, 59.7, 103.6, 111.7, 114.1, 120.3, 123.3, 125.8, 128.1, 132.1, 136.8, 157.6, 161.1, 171.8.
HRMS (ESI): m/z calcd for C15H16ClN4O2S [M - H]- 351.0677, found 351.0681.
3.1.25. Allyl allyl(2-amino-2-(thiazol-2-yl)ethyl)carbamate (13c)
The thiazole (2.99 mL, 3.58 g, 42.1 mmol, 1.5 equiv.) was dissolved in THF (42 mL) and cooled to −78 °C. At this temperature, n-BuLi (2.5 M in hexane, 16.84 mL, 1.5 equiv.) was added dropwise under a nitrogen atmosphere. The reaction mixture was stirred for 20 min at −78 °C, and appropriate R- or S-imine (8.04 g, 28.1 mmol, 1 equiv.) was added dropwise as a solution in THF (1M, 28 mL). The reaction mixture was slowly (1 h) warmed to 0 °C and poured into water (150 mL). The organic layer was separated, and water was extracted with CH2Cl2 (3 × 100 mL). Combined organic phases were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and evaporated to give a brown oil which was purified by flash column chromatography (eluent: hexane-EtOAc, 3:1 → 1:1 → 0:1). This product was used without analysis.
To a solution of the protected compound from the previous step in MeOH (50 ml), 1M solution of HCl in MeOH (100 ml) was added in one portion. The mixture was stirred for 2–3 h. After that, solvent was evaporated, and the residue was dissolved in water (50 mL) and extracted from impurities with CH2Cl2 (2 × 50 mL). After that, solid K2CO3 was added carefully (CO2 evolution!) to pH 10–12. The product was extracted from water with CH2Cl2 (4 × 50 mL), combined organic layers were dried over anhydrous Na2SO4, filtered, and evaporated to give pure amine.
13c(fR): M=3.94 g. Yield (over two steps) = 53%.
13c(fS): M=3.69 g. Yield (over two steps) = 49%.
1H NMR (CDCl3, 400 MHz): δ= 1.92 (br. s., 2 H), 3.49 – 3.96 (m, 4 H), 4.44 – 4.61 (m, 3 H), 5.03 – 5.20 (m, 3 H), 5.26 (d, J=17.24 Hz, 1 H), 5.73 (br. s., 1 H), 5.82 – 5.94 (m, 1 H), 7.24 (d, J=3.30 Hz, 1 H), 7.70 (d, J=3.18 Hz, 1 H).
13C NMR (CDCl3, 100 MHz): δ= (50.6, 50.8), (53.1, 53.2), (53.4, 54.1), 66.3, (116.9, 117.4), (117.5, 117.9), 119.0, 132.8, 133.2, 142.8, 150.8, 175.3.
3.1.26. N-(2-amino-1-(thiazol-2-yl)ethyl)-5-(4-chlorophenyl)-1H-pyrrole-2-carboxamide (13)
Compounds 13(fR), NBD-14150 and 13(fS), NBD-14151 were obtained following the general procedure A and B from amine 13c(fS), 13c(fR), and acid 13d.[14] The resulting compounds were purified using column chromatography on silica gel. Eluent CHCl3-MeOH saturated with NH3 (10:1 and 5:1).
13(fR), NBD-14150: M = 391 mg. Yield = 27% (over two steps). rt = 1.262 min. Purity = 97%. LC-MS: m/z [M+H]+ = 347 Da.
13(fS), NBD-14151: M = 420 mg. Yield = 29% (over two steps). rt = 1.384 min. Purity = 100%. LC-MS: m/z [M+H]+ = 347 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 1.73 (br. s., 2 H), 3.02 (dd, J=13.02, 7.76 Hz, 1 H), 3.16 (dd, J=13.20, 5.38 Hz, 1 H), 5.22 – 5.31 (m, 1 H), 6.65 (d, J=3.55 Hz, 1 H), 7.01 (d, J=3.55 Hz, 1 H), 7.42 (d, J=8.31 Hz, 2 H), 7.58 – 7.63 (m, 1 H), 7.73 – 7.78 (m, 1 H), 7.84 (d, J=8.31 Hz, 2 H), 8.58 (d, J=7.46 Hz, 1 H), 11.85 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 45.7, 54.3, 107.6, 113.0, 119.6, 126.4, 127.4, 128.6, 130.7, 131.1, 133.8, 142.4, 160.5, 172.5.
HRMS (ESI): m/z calcd for C16H16ClN4OS [M + H]- 347.0728, found 347.0725.
3.1.27. N-(2-aminoethyl)-5-(4-chloro-3-fluorophenyl)-1H-pyrrole-2-carboxamide hydrochloride (14)
Compound 14, NBD-14045 was obtained following the general procedure A and Boc-deprotection (below) from tert-butyl (2-aminoethyl)carbamate and acid 13d.[14]
Boc-deprotection: To Boc-protected compound from the last step was added 50 mL of 1 M HCl in methanol. The solution was stirred for 2 h at room temperature, and then the solvent was evaporated. The precipitate was triturated in Et2O (50 mL) and filtered. The product was washed with Et2O (2 × 50 mL) and CH2Cl2 (2 × 50 mL) and dried on the filter.
14, NBD-14045: M = 217 mg. Yield = 33% (over two steps). rt = 1.255 min. Purity = 100%. LC-MS: m/z [M+H]+ = 282 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 2.94 – 3.02 (m, 2 H), 3.45 – 3.54 (m, 2 H), 6.73 (s, 1 H), 6.90 (s, 1 H), 7.55 (t, J=8.2 Hz, 1 H), 7.72 (d, J=8.2 Hz, 1 H), 7.99 (d, J=11.6 Hz, 1 H), 8.04 (br. s., 3 H), 8.62 (t, J=4.8 Hz, 1 H), 12.07 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 36.5, 38.8, 108.5, 112.6 (d, J=22.7 Hz) 113.0, 116.9 (d, J=17.6 Hz), 121.7 (d, J=2.9 Hz), 128.3, 130.8, 133.0 (d, J=7.3 Hz), 132.4, 157.5 (d, J=244.4 Hz), 160.6.
HRMS (ESI): m/z calcd for C13H14ClFN3O [M+H]+ 282.0804, found 282.0804.
3.1.28. 5-(4-chloro-3-fluorophenyl)-N-(piperidin-4-yl)-1H-pyrrole-2-carboxamide (15)
Compound 15, NBD-14094 was obtained following the general procedure A and Boc-deprotection (below) from tert-butyl 4-aminopiperidine-1-carboxylate and acid 13d.[14]
Boc-deprotection: To Boc-protected compound was added 50 mL of 1 M HCl in methanol. The solution was stirred for 2 h at room temperature, and then the solvent was evaporated. Then 10% aqueous K2CO3 solution (50 mL) was added, followed by the addition of CH2Cl2 (100 mL). The layers were separated, and the aqueous layer was extracted with CH2Cl2 (2 × 50 mL). The combined organic phases were dried over anhydrous Na2SO4 and evaporated. The compound was purified using column chromatography on silica gel. Eluent: CHCl3-MeOH saturated with NH3 (10:1).
15, NBD-14094: M = 273 mg. Yield = 64% (over two steps). rt = 1.370 min. Purity = 96%. LC-MS: m/z [M+H]+ = 322 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 1.39 (qd, J=11.8, 3.8 Hz, 2 H), 1.75 (d, J=10.1 Hz, 2 H), 2.50 – 2.61 (m, 2 H), 2.97 (d, J=11.7 Hz, 2 H), 3.74 – 3.90 (m, 1 H), 6.68 (d, J=3.8 Hz, 1 H), 6.88 (d, J=3.8 Hz, 1 H), 7.53 (t, J=8.2 Hz, 1 H), 7.66 (dd, J=8.4, 1.6 Hz, 1 H), 7.84 – 7.97 (m, 2 H), 11.79 (br. s., 1 H). (One exchangeable proton signal is missing)
13C NMR (DMSO-d6, 100 MHz): δ= 33.1, 45.2, 46.7, 108.3, 112.0, 112.4 (d, J=23.0 Hz), 116.8 (d, J=17.9 Hz), 121.6 (d, J=3.3 Hz), 128.8, 130.7, 132.0 (d, J=2.0 Hz), 133.1 (d, J=7.9 Hz), 157.5 (d, J=244.7 Hz), 159.3
HRMS (ESI): m/z calcd for C16H18ClFN3O [M+H]+ 322.1117, found 322.1122.
3.1.29. 5-(4-chloro-3-fluorophenyl)-N-(2,2,6,6-tetramethylpiperidin-4-yl)-1H-pyrrole-2-carboxamide (16)
Compound 16, NBD-14095 was obtained following the general procedure A from 2,2,6,6-tetramethylpiperidin-4-amine and acid 13d.[14] The resulting compound was purified using a column chromatography on silica gel. Eluent: CHCl3-MeOH saturated with NH3 (10:1).
16, NBD-14095: M = 182 mg. Yield = 48%. rt = 1.346 min. Purity = 94%. LC-MS: m/z [M+H]+ = 378 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 1.05 (s, 6 H), 1.12 (t, J=12.3 Hz, 2 H), 1.17 (s, 6 H), 1.70 (dd, J=12.3, 3.6 Hz, 2 H), 3.33 (br. s., 2 H), 4.17 – 4.31 (m, 1 H), 6.68 (d, J=2.9 Hz, 1 H), 6.85 (d, J=3.6 Hz, 1 H), 7.54 (t, J=8.2 Hz, 1 H), 7.65 (dd, J=8.5, 1.8 Hz, 1 H), 7.77 (d, J=7.9 Hz, 1 H), 7.90 (dd, J=11.4, 1.9 Hz, 1 H), 11.73 (br. s., 1 H). (One exchangeable proton signal is missing)
13C NMR (DMSO-d6, 100 MHz): δ= 28.5, 35.0, 42.9, 45.5, 51.4, 108.5 110.5, 113.0 (d, J = 22.7 Hz), 119.5 (d, J = 17.6 Hz), 121.1 (d, J = 3.7 Hz), 127.7, 131.1, 132.5 (d, J = 8.1 Hz), 133.7, 158.5 (d, J = 248.1 Hz), 160.5.
HRMS (ESI) calcd for C20H26ClFN3O [M+H]+ 378.1743, found 378.1736.
3.1.30. Tert-butyl 4-amino-4-cyanopiperidine-1-carboxylate (17b)
To a solution tert-butyl 4-oxopiperidine-1-carboxylate (20.0 g, 101 mmol, 1 equiv.) in MeOH-NH3 (60 mL) and aqueous NH3 (40 mL), NaCN (5.42 g, 111 mmol, 1.1 equiv.) and NH4Cl (6.45 g, 121 mmol, 1.2 equiv.) were added. The mixture was stirred for 4–5 days, then water (200 mL) was added and extracted with DCM (3×150 mL). Combined organic layers were washed with brine (200 mL) and dried over anhydrous Na2SO4, filtered, and concentrated. The obtained crude product was used in the next step without further purification. The compound was obtained in racemic form. M = 21.47 g. Yield = 95%.
1H NMR (CDCl3, 400 MHz): δ= 1.46 (s, 9 H), 1.58 – 1.68 (m, 2 H), 1.86 – 2.01 (m, 4 H), 3.11 – 3.24 (m, 2 H), 3.95 (br. s., 2 H).
13C NMR (CDCl3, 100 MHz): δ= 28.5 (3 C), 31.1, 36.9 (2 C), 50.0 (2 C), 80.3, 123.2, 154.5.
3.1.31. Tert-butyl 4-((tert-butoxycarbonyl)amino)-4-cyanopiperidine-1-carboxylate (17c)
Compound 17b (23.13 g, 103 mmol, 1 equiv.) was dissolved in THF (255 mL), then Boc2O (22.38 g, 103 mmol, 1 equiv.) was added in a few portions. The resulting mixture was stirred at room temperature for 3 days. The solvent was evaporated, and the crude product was purified by flash chromatography on silica gel (eluent: hexane-EtOAc, 5:1 → 1:1). M = 25.39 g. Yield = 76%.
1H NMR (CDCl3, 400 MHz): δ= 1.45 (s, 9 H), 1.48 (s, 9 H), 1.69 – 1.80 (m, 2 H), 2.32 (d, J=12.23 Hz, 2 H), 3.19 – 3.29 (m, 2 H), 3.93 (d, J=13.57 Hz, 2 H), 4.93 (br. s., 1 H).
13C NMR (CDCl3, 100 MHz): δ= 28.2 (3 C), 28.3 (3 C), 34.9 (2 C), 39.8 (br., 2 C), 50.6, 80.2, 81.5 (br.), 119.2, 154.0 (br.), 154.3.
3.1.32. Tert-butyl 4-((tert-butoxycarbonyl)amino)-4-carbamothioylpiperidine-1-carboxylate (17d)
To a solution of appropriate cyanopiperidine 17c (12.35 g, 38.0 mmol, 1 equiv.) in EtOH (380 mL), NaSH trihydrate (41.75 g, 380 mmol, 10 equiv.), and NH4Cl (20.31 g, 380 mmol, 10 equiv.) were added. The resulting mixture was stirred at room temperature for 2 days and then poured into water (1000 mL). The resulting solution was extracted with CH2Cl2 (3 × 200 mL). The combined organic layers were washed with water (200 mL) and brine (200 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness. The crude product was used in the next step without further purification. M = 13.43 g. Yield = 98%.
1H NMR (DMSO-d6, 400 MHz): δ= 1.37 (s, 9 H), 1.38 (s, 9 H), 1.84 – 2.16 (m, 4 H), 2.85 (br. s., 2 H), 3.77 (d, J=8.44 Hz, 2 H), 6.98 (br. s., 1 H), 8.69 (br. s., 1 H), 9.61 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 28.1 (3 C), 28.2 (3 C), 33.2 (br., 2 C), 39.8 (br.,2 C), 61.7, 78.3 (br.), 78.6, 153.6 (br.), 153.8, 154.2.
3.1.33. Ethyl 2-(1-(tert-butoxycarbonyl)-4-(N-(tert-butoxycarbonyl)-2,2,2-trifluoroacetamido)piperidin-4-yl)thiazole-4-carboxylate (17e)
Thioamide 17d (13.43 g, 37.4 mmol, 1 equiv.) was dissolved in THF (374 mL), and to this stirred solution, KHCO3 (29.92 g, 299 mmol, 8 equiv.) and ethyl bromopyruvate (21.9 g, 112 mmol, 3 equiv.) were added at 0 °C. After stirring for 5 min at this temperature, the reaction mixture was warmed to 25 °C, stirred for 30 h, then concentrated. The residue was diluted in CH2Cl2 (200 mL) and washed with H2O (250 mL), brine (250 mL), and dried over anhydrous Na2SO4. After the solution was concentrated, the residue was taken up in THF (250 mL) and cooled to −10 °C. Pyridine (30.1 mL, 374 mmol, 10 equiv.) and trifluoroacetic anhydride (25.96 mL, 187 mmol, 5 equiv.) were slowly added, and the solution was allowed to stir for 2 h at 0 °C. Then the reaction mixture was poured into water (250 mL) and made slightly basic (pH ~ 8) with NaHCO3. The organic layer was separated; water was extracted with CH2Cl2 (3 × 100 mL). Combined organic phases were dried over anhydrous Na2SO4, filtered, and evaporated. The residue was subjected to flash chromatography on silica gel (eluent: hexane-EtOAc, 5:1 → 2:1) to afford thiazole 17e. M = 18.37 g. Yield = 89%.
1H NMR (CDCl3, 400 MHz): δ= 1.38 (t, J=7.15 Hz, 3 H), 1.46 (s, 9 H), 1.51 (s, 9 H), 2.32 – 2.48 (m, 2 H), 2.65 – 2.75 (m, 2 H), 3.34 (t, J=10.64 Hz, 2 H), 3.78 (td, J=9.81, 3.97 Hz, 2 H), 4.38 (q, J=7.09 Hz, 2 H), 8.14 (s, 1 H).
13C NMR (CDCl3, 100 MHz): δ= 14.4, 27.4 (3 C), 28.4 (3 C), 35.5 (2 C), 39.7 (br., 2 C), 61.3, 65.6, 80.0, 87.3, 115.3 (q, J=288.3 Hz), 127.9, 146.6, 150.4, 154.5, 161.1, 164.5 (q, J=39.4 Hz), 173.1.
3.1.34. Methyl 2-(1-(tert-butoxycarbonyl)-4-((tert-butoxycarbonyl)amino)piperidin-4-yl)thiazole-4-carboxylate (17f)
To a solution of 17e (18.37 g, 33.3 mmol) in MeOH (330 mL), a MeONa solution in MeOH (1M, 330 mL) was added. The mixture was stirred at room temperature for 2 h, then poured into saturated NH4Cl solution (500 mL) and extracted with CH2Cl2 (4 × 150 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and evaporated. The crude product was purified by flash chromatography on silica gel (eluent: hexane-EtOAc, 3:1 → 1:1). By treatment with sodium methylate, the corresponding methyl ester was obtained. M = 14.37 g. Yield = 98%.
1H NMR (CDCl3, 400 MHz): δ= 1.39 (br. s., 9 H), 1.45 (s, 9 H), 2.19 – 2.37 (m, 4 H), 3.12 (t, J=12.29 Hz, 2 H), 3.92 (s, 3 H), 3.99 (d, J=13.69 Hz, 2 H), 5.14 (br. s., 1 H), 8.11 (s, 1 H).
13C NMR (CDCl3, 100 MHz): δ= 28.4 (3 C), 28.5 (3 C), 35.6 (br., 2 C), 39.3 (br., 2 C), 52.5, 57.2, 80.0, 80.7 (br.), 127.7, 146.6, 154.4, 154.6, 162.0, 178.1 (br.).
3.1.35. Tert-butyl 4-((tert-butoxycarbonyl)amino)-4-(4-(hydroxymethyl)thiazol-2-yl)piperidine-1-carboxylate (17g)
Compound 17f (14.37 g, 32.5 mmol, 1 equiv.) was dissolved in EtOH (325 mL), then NaBH4 (12.37 g, 325 mmol, 10 equiv.) was carefully added portion-wise. The resulting mixture was stirred at room temperature for 15 min. Then the solution was refluxed for 12–15 h, and the solvent evaporated to half volume and poured into saturated NH4Cl solution (500 mL), and extracted with CH2Cl2 (4 × 150 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and evaporated. The crude product was purified by flash chromatography on silica gel (eluent: hexane-EtOAc, 5:1 → 1:1). M = 3.98 g. Yield = 30%.
1H NMR (CDCl3, 400 MHz): δ= 1.32 (br. s., 9 H), 1.39 (s, 9 H), 1.99 – 2.38 (m, 4 H), 3.05 (br. s., 2 H), 3.86 (d, J=10.15 Hz, 3 H), 4.61 (s, 2 H), 5.43 (br. s., 1 H), 7.04 (s, 1 H).
13C NMR (CDCl3, 100 MHz): δ= 28.3 (3 C), 28.4 (3 C), 35.1 (br.), 36.0 (br.), 38.8 (br.), 39.8 (br.), 56.9, 60.7, 79.7, 80.0 (br.), 114.1, 154.4 (br.), 154.6, 156.3, 177.8.
3.1.36. (2-(4-aminopiperidin-4-yl)thiazol-4-yl)methanol dihydrochloride (17h)
To a solution containing diBoc-protected compound 17g (3.98 g, 9.62 mmol) in MeOH (10 mL), 1M HCl solution in MeOH (50 mL) was added in one portion. The mixture was stirred at room temperature for 4–5 h, and the solvent was evaporated to dryness. The crude product was used in the next step without further purification. The desired product was obtained as a dihydrochloride. M = 2.7 g. Yield = 98%.
1H NMR (DMSO-d6, 400 MHz): δ= 2.36 – 2.47 (m, 2 H), 2.57 – 2.68 (m, 2 H), 2.91 – 3.02 (m, 2 H), 3.45 – 3.57 (m, 2 H), 4.58 (s, 2 H), 7.65 (s, 1 H), 9.36 (br. s., 3 H), 9.45 (br. s., 1 H), 9.79 (br. s., 1 H).
13C NMR (DMSO-d6, 100 MHz): δ= 31.2 (2 C), 39.0 (2 C), 54.5, 59.6, 117.6, 157.7, 167.3.
3.1.37. Tert-butyl 4-amino-4-(4-(hydroxymethyl)thiazol-2-yl)piperidine-1-carboxylate (17i)
Dihydrochloride 17h (2.2 g, 7.68 mmol, 1 equiv.) was dissolved in MeOH (77 mL), then DIPEA (13.4 mL, 76.8 mmol, 10 equiv.) was added in one portion. The mixture was cooled to −40 – −30 °C, and Boc2O (1.84 g, 8.46 mmol, 1.1 equiv.) was added dropwise as a solution in CH2Cl2 (10 mL). The reaction mixture was slowly warmed to room temperature (1–2 h) and stirred for 1 h. The resulting solution was poured into 10% aqueous Na2CO3 (100 mL), and it was extracted with CH2Cl2 (4 × 100 mL). Combined organic layers were dried over anhydrous Na2SO4, filtered, and evaporated. The crude product was purified by flash column chromatography on silica gel (eluent: CH2Cl2-MeOH, 20:1 → 10:1). M = 1.27 g. Yield = 53%.
1H NMR (CDCl3, 400 MHz): δ= 1.46 (s, 9 H), 1.72 (d, J=13.45 Hz, 2 H), 2.07 – 2.17 (m, 2 H), 2.52 (br. s., 3 H), 3.37 (t, J=10.88 Hz, 2 H), 3.71 – 3.86 (m, 2 H), 4.71 (s, 2 H), 7.11 (s, 1 H).
13C NMR (CDCl3, 100 MHz): δ= 28.4 (3 C), 38.1 (2 C), 39.3 (br.), 40.1 (br.), 54.7, 60.6, 79.7, 114.5, 154.7, 156.5, 181.0.
3.1.38. N-(4-(4-(hydroxymethyl)thiazol-2-yl)piperidin-4-yl)-5-(4-(trifluoromethyl)phenyl)-1H-pyrrole-2-carboxamide (17)
DIPEA (0.704 mL, 4.05 mmol, 1 equiv.) was added to an appropriate acid 17k[14] (1.03 g, 4.05 mmol, 1 equiv.) followed by DMF (10 mL) and then HBTU (1.54 g, 4.05 mmol, 1 equiv.). The resulting solution was stirred for 5 min and added to a solution of appropriate amine 17i (1.27 g, 4.05 mmol, 1 equiv.) in DMF (10 mL) in one portion. The reaction mixture was stirred overnight; DMF was evaporated, and the residue was dissolved in CH2Cl2 (100 mL) and successively washed with 5% aqueous NaOH (50 mL) and 10% tartaric acid (50 mL) solutions. The organic layer was dried over anhydrous Na2SO4, filtered, evaporated. The crude product was purified by flash column chromatography on silica gel (eluent: hexane-EtOAc, 3:1 →1:1 → 0:1). (Note that after purification, we detected that product with N- and O-acylation was formed (Rf = 0.3 in hexane-EtOAc, 1:1). It was hydrolyzed with LiOH (10 equiv.) in THF-MeOH-H2O (1:1:1, 21 mL) under reflux for 2 h followed by acidification with aqueous HCl (12M, 10 equiv.)).
To the Boc-protected compound was added 50 mL of 1 M HCl in methanol and allowed 2 h in it. The solvent was evaporated, and 10% aqueous K2CO3 (50 mL) was added, followed by CH2Cl2-EtOH (∼4:1, 50 mL) addition. The organic layer was separated, and the aqueous layer was extracted with CH2Cl2-EtOH (∼4:1, 4 × 50 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by flash chromatography (twice) (eluent 1: CHCl3-MeOH (saturated with NH3∼7M), 10:1 → 5:1, eluent 2: CH2Cl2-MeOH, 2:1 → 1:1) afforded the desired amine as a white solid. 17(NBD-14206): M = 613 mg. Yield = 34% (over two steps). rt = 1.340 min. Purity = 100%. LC-MS: m/z [M+H]+ = 451 Da.
1H NMR (DMSO-d6, 400 MHz): δ= 1.96 – 2.12 (m, 2 H), 2.51 – 2.57 (m, 2 H), 2.77 – 2.91 (m, 4 H), 3.47 (br.s., 1 H), 4.52 (s, 2 H), 5.22 (br. s., 1 H), 6.78 (d, J=3.79 Hz, 1 H), 7.06 (d, J=3.79 Hz, 1 H), 7.23 (s, 1 H), 7.70 (d, J=8.44 Hz, 2 H), 8.03 (d, J=8.19 Hz, 2 H), 8.26 (s, 1 H), 12.01 (br. s., 1 H). (Two exchangeable proton signals are missing)
13C NMR (DMSO-d6, 100 MHz): δ= 35.9 (2 C), 41.6 (2 C), 57.8, 59.9, 108.7, 113.3, 113.6, 124.4 (q, J=272.2 Hz), 125.0 (2 C), 125.6 (q, J=3.7 Hz), 126.6 (q, J=31.5 Hz, 2 C), 128.9, 133.1, 135.6, 156.9, 160.2, 178.2.
HRMS (ESI): m/z calcd for C21H22F3N4O2S [M + H]- 451.1410, found 451.1409.
3.1.39. Tert-butyl 4-(2-((tert-butylsulfinyl)imino)ethyl)piperidine-1-carboxylate (18b)
Tert-butyl 4-(2-oxoethyl)piperidine-1-carboxylate (5.66 g, 25 mmol, 1 equiv.) was dissolved in CH2Cl2 (25 mL) and (S)-(+)-2-methyl-2-propanesulfinamide (3.32 g, 27 mmol, 1.1 equiv.) was added followed by anhydrous CuSO4 (9.94 g, 62 mmol, 2.5 equiv.) addition. The reaction mixture was vigorously stirred for 1 day (at this point, TLC indicated consumption of starting material). Hexane (~100 mL) was added to the reaction mixture, and it was filtered through 1–2 cm plug of silica gel. The precipitate was washed with the hexane-EtOAc mixture (1:1, 2×100 mL), and the filtrate was evaporated. The residue was used without further purification. M(fS) = 7.56 g. Yield = 92%.
Reaction with (R)-(+)-2-methyl-2-propanesulfinamide was performed in the same way. M(fR) = 7.73 g. Yield = 92%.
1H NMR: (CDCl3, 400 MHz) δ = 1.09 – 1.30 (m, 2H), 1.17 (s, 9 H), 1.43 (s, 9 H), 1.64 – 1.73 (m, 2 H), 1.80 – 1.97 (m, 1 H), 2.45 (t, J=5.4 Hz, 2 H), 2.69 (t, J=10.3 Hz, 2 H), 3.87 – 4.31 (m, 2 H), 8.03 (t, J=5.0 Hz, 1 H).
13C NMR: (CDCl3, 100 MHz) δ = 22.5, 28.5, 32.1, 33.7, 42.8, 43.9 (br.), 56.7, 79.5, 154.8, 168.3.
3.1.40. (2-(1-amino-2-(piperidin-4-yl)ethyl)thiazol-4-yl)methanol trihydrochloride (18c) Experimental procedures are given for the synthesis of R-enantiomer (fR):
The appropriate thiazole (5.90 g, 25.7 mmol, 1.1 equiv.) was dissolved in THF (26 mL). Under a nitrogen atmosphere, the solution was cooled to −78 °C, and at this temperature n-BuLi (2.5M in hexane, 11.0 mL, 27.5 mmol, 1.2 equiv.) was added dropwise. The reaction mixture was stirred for 10 min, and a solution of imine 18b(fR) (7.73 g, 23.4 mmol, 1 equiv.) in THF (26 mL) was added dropwise. The resulting mixture was allowed to warm to room temperature, stirred for one h, and diluted with water (~200 mL). The product was extracted with CH2Cl2 (3×100 mL). Combined organic layers were dried over Na2SO4, filtered, and evaporated. The residue was purified using flash chromatography on silica gel (eluent: hexane-EtOAc 3:1 and pure EtOAc).
To a solution of protected thiazole from the previous step in MeOH (50 ml), 1M solution of HCl in MeOH (100 ml) was added in one portion. The mixture was stirred for 2–3 h, and the solvent was evaporated. The residue was recrystallized from methanol (~ 20 mL). The precipitate was filtered, quickly washed with 50 mL of Et2O-methanol (2:1) mixture, and quickly transferred under vacuum (the product is hygroscopic). M = 4.86 g. Yield (over two steps) = 59%.
S-enantiomer (fS) was synthesized by the same experimental procedures. M = 5.25 g. Yield (over two steps) = 64%.
1H NMR: (DMSO-d6, 400 MHz) δ = 1.29 – 1.47 (m, 2 H), 1.50 – 1.64 (m, 1 H), 1.74 (d, J=13.2 Hz, 1 H), 1.77 – 1.90 (m, 2 H), 1.96 (dt, J=13.6, 6.8 Hz, 1 H), 2.69 (td, J=22.1, 11.5 Hz, 2 H), 3.14 (dd, J=22.3, 12.3 Hz, 2 H), 4.56 (s, 2 H), 4.72 (d, J=5.0 Hz, 1 H), 7.51 (s, 1 H), 8.97 (br. s., 5 H), 9.08 – 9.21 (m, 1 H), 9.21 – 9.33 (m, 1 H).
13C NMR: (DMSO-d6, 100 MHz) δ = 27.7, 28.0, 29.6, 40.1, 42.7, 42.7, 48.9, 59.6, 116.5, 157.9, 165.5.
3.1.41. N-(1-(4-(hydroxymethyl)thiazol-2-yl)-2-(piperidin-4-yl)ethyl)-5-(4-(trifluoromethyl)phenyl)-1H-pyrrole-2-carboxamide (18)
To a solution of salt 18c (fS or fR) (1.00 g, 2.85 mmol, 1 equiv.) in DMF (10 mL), DIPEA (1.49 mL, 8.55 mmol, 3 equiv) was added. The solution of Boc2O (622 mg, 2.85 mmol, 1 equiv.) in DMF (5 mL) was added dropwise. After 1 h in another flask DIPEA (0.497 mL, 2.85 mmol, 1 equiv.) was added to an acid 17k[14] (728 mg, 2.85 mmol, 1equiv.) followed by DMF (8 mL) and then HBTU (1.081 g, 2.85 mmol, 1 equiv.). A solution of activated acid was added to a solution of protected amine. The reaction mixture was stirred overnight. Then DMF was evaporated, and the residue was dissolved in DCM (50 mL) and successively washed with 5% aqueous NaOH and 10% tartaric acid or citric acid aqueous solutions (50 mL). The organic layer was dried over Na2SO4, filtered, evaporated, and dry loaded on silica. Eluting with pure EtOAc gave protected compounds.
To Boc-protected compound was added 30 mL of 1 M HCl in methanol. After 2 h, methanol was evaporated, and 10% aqueous K2CO3 (50 mL) was added, followed by CH2Cl2-EtOH (∼4:1, 50 mL) addition. The organic layer was separated, and the aqueous layer was extracted with CH2Cl2-EtOH (∼4:1, 4 × 50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. Purification by flash chromatography (twice) (eluent 1: CHCl3-MeOH (saturated with NH3∼7M), 10:1 and 5:1, eluent 2: CH2Cl2-MeOH, 4:1 and 2:1) afforded amine as a white solid.
18(fR), NBD-14230: M = 358 mg. Yield = 26% (over three steps). rt = 1.444 min. Purity = 100%. LC-MS: m/z [M+H]+ = 479 Da.
18(fS), NBD-14228: M = 554 mg. Yield = 41% (over three steps). rt = 1.429 min. Purity = 100%. LC-MS: m/z [M+H]+ = 479 Da.
1H NMR: (DMSO, 400 MHz) δ = 1.06 – 1.23 (m, 2 H), 1.43 – 1.53 (m, 1 H), 1.57 (d, J=12.7 Hz, 1 H), 1.71 (d, J=11.9 Hz, 1 H), 1.93 (t, J=7.3 Hz, 2 H), 2.26 – 2.46 (m, 2 H), 2.90 (d, J=11.7 Hz, 2 H), 4.53 (s, 2 H), 5.42 (dd, J=15.3, 8.4 Hz, 1 H), 6.77 (d, J=3.8 Hz, 1 H), 7.03 (d, J=3.8 Hz, 1 H), 7.27 (s, 1 H), 7.69 (d, J=8.4 Hz, 2 H), 8.03 (d, J=8.2 Hz, 2 H), 8.79 (d, J=8.6 Hz, 1 H), 12.13 (br. s., 1 H). (Two proton signals missing due to proton exchanging phenomena)
13C NMR: (DMSO, 100 MHz) δ = 31.7, 32.6, 33.3, 41.5, 45.8, 45.9, 48.1, 59.8, 108.9, 113.1, 114.0, 124.4 (q, J = 271.6 Hz), 125.1 (2C), 125.6 (q, J = 3.7 Hz, 2C), 126.7 (q, J = 31.7 Hz), 128.2, 133.4, 135.6, 157.7, 160.1, 174.2.
HRMS (ESI): m/z calcd for C23H26F3N4O2S [M+H]+ 479.1723, found 479.1728.
4.0. Biology Methods
The antiviral activity of the NBD compounds was evaluated in a single-cycle infection assay by infecting TZM-bl cells with the HIV-1 NL4–3-HXB2-Luc pseudotyped virus expressing Env of the lab-adapted HIV-1HXB-2 (CXCR4) and the clinical isolate HIV-1WEAUd15.410.5017 (NIH # 11578) (dual-tropic CCR5/CXCR4) as previously described[11]. The infection of U87-CD4-CCR5 cells with the control pseudovirus VSV-G, and the assay in CD4-negative Cf2Th-CCR5 cells infected with the luciferase-expressing recombinant CD4-dependent pseudovirus HIV-1ADA were performed as previously described[11, 12, 24]. The cytotoxicity of the small molecules in TZM-bl, U87-CD4-CCR5 and Cf2Th-CCR5 cells was measured by the colorimetric XTT method, as previously described[8, 15]. The activity of the NBD compounds against the HIV-1 Reverse Transcriptase (RT) was evaluated by using the Colorimetric Reverse Transcriptase Assay (Roche) and following the manufacturer’s instructions.
5.0. Conclusions
We were successful in validating the synthetic procedure for new scaffolds based on NBD-14136 amine and acid intermediate by isosteric replacements and were able to profile a couple of compounds from each new scaffold with antiviral potency and cell toxicity. Among them, the compounds 12, 13, and 17 resulted as leads for further follow-up and chemical optimization to convert to more potent inhibitors. While they were less active than NBD-14136 or NBD-14091 and NBD-14092, they showed reasonable antiviral activity and cytotoxicity profile. However, due to the lack of specificity in antiviral activity, 17 could not be considered one of the best leads. Notably, compound 12 was almost equipollent in terms of antiviral activity to the reference compounds NBD-14091 and NBD-14092. It demonstrated a two-fold improvement of cytotoxicity, indicating that position 6 of the indole could be an interesting site for additional functionalizations. Most of all, the overall platform remains an exciting, drug-like, and diverse tool for the exploration and initial probing of any potential target suitable for small molecule drug discovery projects. These compounds are diverse and conform to drug-like criteria[19, 33, 34] and demonstrated to have a reasonable solubility for the biological assay (they have been tested in DMSO/PBF buffers). Moreover, all the key intermediates have been developed and validated in large quantities, rendering them less problematic for any potential chemistry follow-up or chemical space expansion.
Supplementary Material
Highlights.
Novel scaffolds as HIV-1 inhibitors targeted to gp120
Novel chemistry for drug-like scaffolds and drug design
Acknowledgments
This study was supported by funds from NIH Grant R01 AI104416 (AKD), and intramural funds from the New York Blood Center (AKD).
Abbreviations
- HIV
Human Immuno Deficiency Virus
- NDMBA
N-Nitrosodimethylamine
- Pd(dba)2
Bis(dibenzylideneacetone)palladium
- Ro3
Rule of 3
- FBDD
Fragment-Based Drug discovery
Footnotes
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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