Preclinical Optimization of gp120 Entry Antagonists as anti-HIV‑1 Agents with Improved Cytotoxicity and ADME Properties through Rational Design, Synthesis, and Antiviral Evaluation

Abstract

We previously reported a milestone in the optimization of NBD-11021, an HIV-1 gp120 antagonist, by developing a new and novel analogue, NBD-14189 (Ref1), which showed antiviral activity against HIV-1_HXB2, with a half maximal inhibitory concentration of 89 nM. However, cytotoxicity remained high, and the absorption, distribution, metabolism, and excretion (ADME) data showed relatively poor aqueous solubility. To optimize these properties, we replaced the phenyl ring in the compound with a pyridine ring and synthesized a set of 48 novel compounds. One of the new analogues, NBD-14270 (8), showed a marked improvement in cytotoxicity, with 3-fold and 58-fold improvements in selectivity index value compared with that of Ref1 and NBD-11021, respectively. Furthermore, the in vitro ADME data clearly showed improvements in aqueous solubility and other properties compared with those for Ref1. The data for 8 indicated that the pyridine scaffold is a good bioisostere for phenyl, allowing the further optimization of this molecule.

Graphical Abstract

INTRODUCTION

According to the Joint United Nations Programme on HIVAIDS (UNAIDS) report, published in 2018, 37 million people are living with HIV, and almost 2 million new infections are diagnosed each year. Despite the availability of more than 30 Food and Drug Administration (FDA)-approved drugs for acquired immunodeficiency syndrome (AIDS) treatment, AIDS remains a major global public health concern, although combination antiretroviral therapies (cART) can help manage this devastating disease. However, no vaccine has yet been developed to manage the transmission of the human immunodeficiency virus (HIV), and despite many years of effort, no cure has been identified for HIV. Therefore, drug-based treatments remain the only option for those who have been infected with the virus. Furthermore, HIV patients must engage in lifelong therapy regimens. Unfortunately, because of the very high heterogeneity among HIV strains, drug-associated toxicities and difficulties with adherence often result in treatment failure. Long-term use of HIV treatment drugs may also lead to the emergence of drug-resistant HIV. As a result, treatment-experienced patients may exhaust their treatment options. Therefore, the development of novel drugs, with fewer side effects and high antiviral potencies against a large array of drug-resistant HIV-1, is urgently needed. To achieve this goal, the continued discovery of new classes of drugs that target novel HIV components is critical.

HIV fusion and attachment (often collectively termed as “entry”) are the most critical steps necessary for HIV to initiate its life cycle in the host cell.^1–4 Despite the importance of these steps, only two FDA-approved drugs target the HIV-1 entry pathway: Fuzeone (Enfuvirtide), which targets HIV-1 glycoprotein 41 (gp41), and Maraviroc, which targets C–C chemokine receptor type 5 (CCR5) coreceptors. Thus far, no FDA-approved drugs target the HIV-1 envelope (Env) glycoprotein gp120. However, the potential of gp120 as a drug target was validated by reports describing several highly potent HIV-1 attachment inhibitors, from Bristol-Meyer Squibs (BMS),^5–13 our group, and others.^14–18 One of the BMS attachment inhibitors, known as Fostemsavir (BMS-663068), is currently in an advanced stage of clinical development and demonstrated success in a recent Phase III clinical trial (Bright Study). Until recently, the target site of this class of attachment inhibitors being developed by BMS was uncertain. In 2017, Pancera et al. reported first the X-ray crystal structures of these inhibitors, with a trimeric HIV-1 envelope.¹⁹ Interestingly, these crystal structures showed that the binding pocket for this class of inhibitors was distinct from the Phe43 cavity induced by the cluster of differentiation 4 (CD4), the cellular receptor that binds to gp120. In contrast, Madani et al. first suggested that NBD-556, which our group identified and reported in 2005,¹³ binds to the highly conserved Phe43 cavity.²⁰ In 2012, we solved the X-ray crystal structure of NBD-556 bound to gp120, confirming that NBD-556 binds to the Phe43 cavity.¹¹ Unfortunately, NBD-556 was shown to be a CD4-agonist that could enhance HIV-1 infection in CD4-CCR5+ cells.²⁰ This undesirable trait motivated us to use the insights gained from the crystal structure to modify the structure of NBD-556, to convert this compound into a gp120 entry antagonist. We were successful and reported the first gp120 entry antagonist, NBD-11021, in 2012.²¹ Subsequently, a series of X-ray structures, showing gp120 entry antagonists bound to monomeric gp120, have confirmed that these antagonists also bind to the Phe43 cavity.^9,11,12,22

Recently, we reported the successful design of our most advanced gp120 antagonist, NBD-14189 (Ref1, Figure 1), which was tested against a large panel of HIV-1 Env-pseudotyped viruses, representing a diverse set of clinical isolates and multiple HIV subtypes.¹⁰ Ref1 showed remarkable antiviral potency (as low as 63 nM), indicating that this compound has broad-spectrum inhibitory activity against HIV-1.¹⁰ Furthermore, this inhibitor had absorption, distribution, metabolism, and excretion (ADME) properties comparable to BMS-626529, a prodrug of which, BMS-663068, is currently undergoing Phase III clinical trials.¹⁰ However, the in vitro ADME and cellular toxicity data confirmed that further improvements could be made to the cellular toxicity of Ref1, to improve the selectivity index (SI = CC₅₀/IC₅₀) (CC₅₀ = 50% cytotoxic concentration, IC₅₀ = half maximal inhibitory concentration) and to the ADME properties, especially, aqueous solubility. Toward that goal, we replaced the phenyl group in Region I (Figure 1) with a pyridine moiety, a unique aromatic ring. The use of pyridine as a bioisostere of aromatic rings has been reported in the fields of medicinal chemistry and drug discovery to improve aqueous solubility, stability, and the ability to form H-bonds.^23–25 Here, we report the detailed synthesis, structure–activity relationships (SARs), antiviral activities, and ADME properties of a large set of pyridine-based analogues, with considerable improvement over the most potent gp120 entry antagonists previously reported by our group. This study explored a new scaffold in Region I that resulted in many improved properties while still displaying potent antiviral activity.

Figure 1.

Chronology of improvement of anti-HIV-1 activity (IC₅₀ against HIV-1HXB2) and SI.

CHEMISTRY

The syntheses of the novel inhibitors are described in Schemes 1–7. First, we prepared a series of 5-arylpyrrole-2-carboxylic acids with pyridine pharmacophoric fragment or its bioisost—pyrimidine and pyridazine (Scheme 1). Acids S4a–S4h were prepared using the general scheme involving Suzuki coupling of pyridine/pyrimidine/pyridazine halogenides with N-Boc-2-pyrrole boronic acid, followed by Boc cleavage, acylation, and semihaloform reaction.²⁶ Aryl bromides and aryl chlorides were commercially available. Acid S8 was prepared using a general procedure A involving Suzuki coupling of 4-bromo-2-fluoropyridine and with N-Boc-2-pyrrole boronic acid, followed by Boc cleavage (MeOH-HCl) afforded the 2-methoxy-4-(1H-pyrrol-2-yl)pyridine S6 in good yield (67%), which was then subjected first to an acylation and then to a semihaloform reaction.^8,21,27–29

Scheme 1.

Synthesis of Intermediate Acids

Scheme 7.

Synthesis of HIV-1 Inhibitors 1–48 in Table 1

5-Aryl-3-methyl-1H-pyrrole-2-carboxylic acids were prepared using commercially available reagents (2-bromo-5-(trifluoromethyl)pyridine or 2-bromo-5-chloropyridine) and known compound N-Boc-β-iododehydroamino acid methyl esters R4 as per the reported procedure²⁹ (Scheme 3). This one-pot, two-step procedure occurs by a Sonogashira coupling followed by a 5-endo-dig-cyclization, which involves the nitrogen atom of the dehydroamino acid.

Scheme 3. Synthesis of 5-(5-Substituted-2-yl)-3-methyl-1H-pyrrole-2-carboxylic Acid and Na Salt^a.

^aAsterisk (*) indicates acidification step performed only for S16.

The Suzuki approach could not be used with the methylpyrrole intermediates S15 and S16 due to chemical feasibility issues related to the synthesis of the methyl pyrrole boc-protected derivative needed as a substrate. Except for S23, S32, and S37, all the required intermediary protected secondary amines were prepared using an enantioselective pathway reported previously.⁹ Instead, S23 and S32, due to the extended and different side chains, were prepared accordingly to Schemes 4 and 5, respectively, but still exploiting the enantioselective step as per the General Procedure E. While the synthetic approach for S37 is depicted in Scheme 6, it was obtained as a racemate. Since the corresponding final compound 31 showed no improvement in anti-HIV-1 activity but, rather, a loss of activity, no attempts were wasted in the isolation of the corresponding enantiomers.

Scheme 4. Synthesis of Allyl Allyl(2-amino-2-(4-(2-((tert-Butyldiphenylsilyl)oxy)-1-hydroxyethyl)thiazol-2-yl)ethyl)carbamate (S23 fS and S23 fR)^a.

^aNote all enantiomer compounds derived from the stereo-controlled addition of the allyl N-allyl-N-[(2E)-2-tert-butylsulfinyliminoethyl]carbamate with absolute configuration S, were specified with the fS descriptor, and all enantiomer compounds derived from the stereo-controlled addition of the allyl N-allyl-N-[(2E)-2-tert-butylsulfinyliminoethyl]carbamate with the absolute configuration R have been specified with the fR descriptor as per previous works.¹⁰

Scheme 5.

Synthesis of Allyl N-Allyl-N-[2-amino-2-[4-[2-[tert-butyl(diphenyl)silyl]oxy-1-hydroxy-ethyl]thiazol-2-yl]ethyl]carbamate (S32 fR and S32 fS)

Scheme 6.

Synthesis of tert-Butyl (2-amino-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)thiazol-2-yl)propyl)carbamate (S37)

The synthesis of all final 48 pyridine analogues (1–48, Table 1) was performed as per Scheme 7. The amines S38–S40 were prepared using a method described in our earlier work. Similarly, amines S41–S45 were synthesized, starting from the appropriate thiazole derivative and following the protocols as reported earlier.^10,28

Table 1.

Anti-HIV-1 Activity (IC50) and Cytotoxicity (CC50) of gp120 Entry Antagonists in Single-Cycle (TZM-bl Cells) Assay

													μMa
No. (enantiomer)	R1	R2	R3	R4	R5	R6	R7	R8	X	Y	Z	W	IC50	CC50
Ref1 (S)	CH2OH	H	H	H	H	H	CF3	F	CH	CH	CH	CH	0.089 ± 0.001	21.9 ± 0.5
Ref2 (R)	H	CH2OH	H	H	H	H	CF3	H	CH	CH	CH	CH	0.27 ± 0.02	42.4 ±1.0
1 (R)	H	CH2OH	H	H	H	H	Cl	H	N	CH	CH	CH	0.85 ± 0.1	144 ± 7.5
2 (S)	H	CH2OH	H	H	H	H	Cl	H	N	CH	CH	CH	2.7 ± 0.7	142 ± 1.7
3 (R)	H	CH2OH	H	H	H	CH3	Cl	H	N	CH	CH	CH	1.2 ± 0.1	98.3 ± 4
4(S)	H	CH2OH	H	H	H	CH3	Cl	H	N	CH	CH	CH	2.4 ± 0.2	95 ± 3.6
5 (R)	H	CH2OH	H	H	H	H	CF3	H	N	CH	CH	CH	0.96 ± 0.1	>122
6 (S)	H	CH2OH	H	H	H	H	CF3	H	N	CH	CH	CH	1.2 ± 0.3	>122
7 (R)	H	CH2OH	H	H	H	CH3	CF3	H	N	CH	CH	CH	0.36 ± 0.01	92.8 ± 2.4
8 (S)	H	CH2OH	H	H	H	CH3	CF3	H	N	CH	CH	CH	0.16 ± 0.004	109.3 ± 2
9 (R)	H	CH2OH	H	CH3	CH3	H	CF3	H	N	CH	CH	CH	0.52 ± 0.09	74 ± 3.6
10 (S)	H	CH2OH	H	CH3	CH3	H	CF3	H	N	CH	CH	CH	0.5 ± 0.09	42.4 ± 4.1
11 (R)	H	CH2OH	H	CH3	CH3	CH3	CF3	H	N	CH	CH	CH	0.6 ± 0.2	35.3 ± 2
12 (S)	H	CH2OH	H	CH3	CH3	CH3	CF3	H	N	CH	CH	CH	0.43 ± 0.05	37.5 ± 2.2
13 (R)	H	CHOHCH2OH	H	H	H	ch3	CH3	H	N	CH	CH	CH	2.1 ± 0.3	>113
14 (S)	H	CHOHCH2OH	H	H	H	ch3	CH3	H	N	CH	CH	CH	5.9 ± 0.6	>113
IS (R)	CH2OH	H	H	H	H	H	Cl	H	N	CH	CH	CH	5 ± 0.5	>132
16 (S)	CH2OH	H	H	H	H	H	Cl	H	N	CH	CH	CH	1.7 ± 0.3	>132
17 (R)	CH2OH	H	H	H	H	H	F	H	N	CH	CH	CH	>11	>138
18 (S)	CH2OH	H	H	H	H	H	F	H	N	CH	CH	CH	>11	>138
19 (R)	CH2OH	H	H	H	H	H	CH3	H	N	CH	CH	CH	9.6 ±1.2	>140
20 (S)	CH2OH	H	H	H	H	H	CH3	H	N	CH	CH	CH	>11	>140
21 (R)	CH2OH	H	H	H	H	H	CF3	H	N	CH	CH	CH	0.59 ± 0.07	94.3 ± 8.6
22 (S)	CH2OH	H	H	H	H	H	CF3	H	N	CH	CH	CH	0.3 ± 0.04	77.9 ± 9.9
23 (R)	CH2OH	H	H	CH3	CH3	H	CF3	H	N	CH	CH	CH	1.7 ± 0.04	61 ± 6.5
24 (S)	CH2OH	H	H	CH3	CH3	H	CF3	H	N	CH	CH	CH	0.58 ± 0.05	59 ± 10
25 (R)	(CH2)2OH	H	H	H	H	H	CF3	H	N	CH	CH	CH	1.7 ± 0.1	100 ± 7
26 (S)	(CH2)2OH	H	H	H	H	H	CF3	H	N	CH	CH	CH	0.76 ± 0.04	91 ± 3.6
27 (R)	(CH2)3OH	H	H	H	H	H	CF3	H	N	CH	CH	CH	2.3 ± 0.3	>114
28 (S)	(CH2)3OH	H	H	H	H	H	CF3	H	N	CH	CH	CH	0.89 ± 0.12	81.2 ± 1.3
29 (R)	CHOHCH2OH	H	H	H	H	H	CF3	H	N	CH	CH	CH	5.2 ± 0.9	>113
30 (S)	CHOHCH2OH	H	H	H	H	H	CF3	H	N	CH	CH	CH	3.8 ± 0.2	>113
31-rac	CH2OH	H	CH3	H	H	CH3	CF3	H	N	CH	CH	CH	2.6 ± 0.4	>68.3
32-rac	CH2OH	H	CH3	H	H	H	CF3	H	N	CH	CH	CH	5.1 ± 0.4	>70.5
33 (R)	CH2OH	H	H	H	H	CH3	CF3	H	N	CH	CH	CH	0.35 ± 0.02	85 ± 3
34 (S)	CH2OH	H	H	H	H	CH3	CF3	H	N	CH	CH	CH	1.1 ± 0.1	85 ± 4
35 (R)	CH2OH	CH2OH	H	H	H	H	CF3	H	N	CH	CH	CH	1.8 ± 0.3	>113
36 (S)	CH2OH	CH2OH	H	H	H	H	CF3	H	N	CH	CH	CH	>13	>113
37 (R)	H	H	H	H	H	H	CF3	H	N	CH	CH	CH	2 ± 0.2	89.3 ± 1
38 (S)	H	H	H	H	H	H	CF3	H	N	CH	CH	CH	1.2 ± 0.03	93.6 ± 1.5
39 (R)	CH2OH	H	H	H	H	H	Cl	H	CH	N	CH	CH	>11	>132
40 (S)	CH2OH	H	H	H	H	H	Cl	H	CH	N	CH	CH	>11	>132
41 (R)	CH2OH	H	H	H	H	H	CF3	H	CH	N	CH	CH	12.7 ± 2.8	>122
42 (S)	CH2OH	H	H	H	H	H	CF3	H	CH	N	CH	CH	11.9 ± 1.9	>122
43 (R)	CH2OH	H	H	H	H	H	CF3	H	N	N	CH	CH	>12	>73
44 (S)	CH2OH	H	H	H	H	H	CF3	H	N	N	CH	CH	>12	>73
45 (R)	CH2OH	H	H	H	H	H	Cl	H	N	CH	N	CH	>15.8	>66
46 (S)	CH2OH	H	H	H	H	H	Cl	H	N	CH	N	CH	>15.8	>66
47 (R)	CH2OH	H	H	H	H	H		O CH3	CH	CH	CH	N	>11	>133
48 (S)	CH2OH	H	H	H	H	H		O CH3	CH	CH	CH	N	>11	>133
BMS-626529													<1 nM	>100

^aThe reported IC50 and CC50 values represent the means ± standard deviations (n = 3).

All the alloc protected intermediates were worked up as per the general procedure H,¹⁰ and the corresponding alloc products (1a–48a) were used directly in the next step without characterization.

RESULTS AND DISCUSSION

Optimization Strategy.

In recent years, we have made significant progress converting NBD-556, the first-in-class CD4 agonist, into gp120 entry antagonists, such as NBD-11021, NBD-14010, and Ref1, while simultaneously dramatically improving the anti-HIV potencies of these new molecules (Figure 1).^9,10,21 Despite this success, we realized that our most potent inhibitor, Ref1, still requires further improvements in cytotoxicity and SI values. Furthermore, the in vitro ADME study indicated that Ref1 requires improved solubility.¹⁰ Poor aqueous solubility can lead to slow drug absorption, which may result in the poor bioavailability of drugs. Therefore, we reasoned that improved aqueous solubility and reduced toxicity are urgently necessary for our best inhibitors. However, the X-ray crystal structures of CD4 bound to gp120 and those of Phe43 cavity-targeted inhibitors bound to gp120 have confirmed that the hydrophobic cavity where Phe43 binds is narrow;^9,22 therefore, the structural manipulation of the phenyl ring in our best inhibitors required careful consideration. We used a pyridine scaffold as a bioisostere, replacing the phenyl ring, due to its aromatic characteristics and increased basicity compared with the phenyl ring, to improve aqueous solubility, minimize reactive metabolite formation, and mitigate toxicity liability.^23–25 While this article was in preparation, Kobayakawa et al. reported successful improvements in aqueous solubility and cytotoxicity by replacing the phenyl ring of NBD-556 with a pyridine scaffold.³⁰

Anti-HIV-1 Screening and Structure–Activity Relationships (SARs).

Our previously reported data for Ref1, the best gp120 entry antagonist that we have identified, showed an anti-HIV-1 activity of 89 nM against HIV-1_HXB2; however, Ref1 showed high cytotoxicity, with an SI value of 246.¹⁰ Furthermore, although the SI value was considered to be reasonable, the cytotoxicity and ADME data indicated that further improvements in cytotoxicity and ADME properties, especially aqueous solubility, were necessary to develop this molecule into a preclinical candidate for further assessment. Until recently, our group and others have avoided altering the phenyl group in Region I (Figure 1), because all of the X-ray crystal structures of this class of compounds bound to HIV-1 gp120 have confirmed that this hydrophobic ring was inserted deep inside the Phe43 cavity, which is surrounded by hydrophobic residues. Pyridine was used by BMS as a substitute for the phenyl ring in the inhibitor that prevents the attachment of HIV gp120 to CD4 host cells, which led to the development of the potent clinical candidate BMS-488043.²⁴ BMS-488043 showed a better pharmacokinetic profile, including improved solubility, and better metabolic profiles than previously described inhibitors. To explore other alternative bioisosteres, we opted to utilize a pyridine scaffold for replacing the phenyl ring (Figure 1).

We made a concerted effort to synthesize pyridine analogues for some of our most active phenyl-containing inhibitors, produced 48 novel pyridine-based compounds, and determined comprehensive SARs for these compounds. In general, we observed that cytotoxicity improved considerably for these compounds compared with Ref1 and a structurally similar analogue, NBD-14136¹⁰ (Ref2, Figure 1), irrespective of the position of “N” within the pyridine ring. The introduction of CH₂OH or higher congeners at position R₁ (15–34) improved cytotoxicity, but antiviral activity was dependent on other substituents in Regions I, II, and III. For example, the introduction of the CH₃ group at positions R₄ and R₅ in Region III generally retained antiviral potency, but the toxicities of these compounds were higher (9–12, 23, and 24). An electron-withdrawing substituent at position R₇ generally improved or maintained antiviral potency; however, antiviral activity reduced considerably when an electron-donating substituent, such as CH₃, was introduced at position R₇ (19 and 20).

Although fluorine is an electron-withdrawing substituent and is hydrophobic, its presence at R₇ reduced antiviral activity, most likely due to its small size. When the N atom in pyridine was at positions Y or W (see the structures in Table 1), the antiviral potencies of these compounds dropped dramatically. Similarly, the presence of a bulkier group at position R₈ also had detrimental effects on antiviral activity, most likely due to steric limitations in the narrow hydrophobic cavity.

Interestingly, the presence of a CH₃ group at position R₆ improved antiviral activity, with no detrimental effects on toxicity. We also introduced a branched alcohol group (CHOHCH₂OH) at position R₁, which resulted in the loss of antiviral activity (13, 14 and 29, 30), although the cytotoxicity of these compounds remained low (higher CC₅₀ values). Similarly, we introduced longer alcohol groups, such as (CH₂)₂OH (25, 26) and (CH₂)₃OH (27, 28) at R₁, and observed no substantial reductions in antiviral activity; however, cytotoxicity was somewhat higher for some of these compounds (25, 26, and 28).

The most notable improvements we observed were for NBD-14270 (8), with an IC₅₀ of 0.16 μM and a CC₅₀ of 109.3 μM. The IC₅₀ of 8 reduced by approximately twofold compared with that of Ref1, while the cytotoxicity (CC₅₀) improved by approximately fivefold. However, the activity and cytotoxicity of 8 improved by 2-fold and 2.5-fold, respectively, compared with those for Ref2. The SI value for Ref1 was 246, whereas the SI value for 8 reached 683, an almost threefold improvement. This remarkable improvement in the cytotoxicity and SI values prompted more in-depth assessments of the antiviral potency and ADME properties of this inhibitor.

Kobayakawa et al., in their recent report describing the replacement of the phenyl ring with pyridine, also showed measurable improvements in cytotoxicity for their pyridine analogues of NBD-556.³⁰ However, the closest analogue of NBD-556 (5-chloro-2-substituted pyridine) showed an antiviral potency (IC₅₀) reduction of approximately 358-fold, whereas the cytotoxicity (CC₅₀) improved by approximately threefold. Besides, some other reported analogues, such as 2-chloro-4-substituted pyridine and 2-chloro-5-substituted pyridine, showed antiviral potency reductions of 13-fold and 23-fold, respectively, whereas the cytotoxicity improvements were also approximately threefold for these compounds.³⁰

The New Generation of Inhibitors Showed Entry Antagonist Traits.

Our first-generation NBD compound, NBD-556,¹³ was reported to facilitate HIV infection into CD4-negative cells that expressed the coreceptor CCR5 by mimicking CD4 and inducing a conformational change in gp120, promoting CCR5 binding and enhancing HIV-1 entry.^31,32 Subsequently, we verified that our new inhibitors did not possess this undesirable trait and behaved as entry antagonists.^9,10 In this study, we initially tested all 48 pyridine-containing molecules in a single-cycle assay against HIV-1_HXB2. We selected 8, which exhibited the best anti-HIV-1 activity and a higher SI value (SI = 683), and NBD-14235 (33), which has an SI of 243 and close structural similarity to 8, to confirm that they are HIV-1 gp120 entry antagonists. CD4-negative and CCR5-positive Cf2TH–CCR5 cells were infected with the recombinant CD4-dependent HIV-1_ADA virus, in the presence of escalating concentrations of these inhibitors. NBD-556 was used as a control. As shown in Figure 2, NBD-556 significantly enabled the infection of the Cf2Th-CCR5 cells, whereas 8 and 33 did not, indicating that these compounds maintained the HIV-1 entry antagonist property. Additionally, these compounds did not show toxicity at the doses used in this assay. We used these two molecules for further antiviral evaluation.

Figure 2.

Infectivity of Cf2Th–CCR5 cells by CD4-dependent HIV-1_ADA. Cf2Th–CCR5 cells were infected with CD4-dependent HIV-1_ADA in the presence of 8 and 33. NBD-556 was 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 greater than 60; for 8 and 33 it was greater than 47. All the values represent the mean ± standard deviation.

Antiviral Activities of 8 and 33 against a Large and Diverse Panel of HIV-1 Env-pseudotyped Clinical Isolates.

We previously reported systematic improvements in the anti-HIV-1 activities of our gp120 entry antagonists against a large panel of diverse HIV-1 Env-pseudotyped clinical isolates.¹⁰ Some of our best inhibitors against HIV-1_HXB2 showed broad-spectrum antiviral activities against these clinical isolates.^9,10,21,27 We selected 8 and 33 to evaluate the anti-HIV-1 activity against a set of 50 HIV-1 clinical isolates belonging to diverse subtypes, including primary, transmitted, and early founder HIV-1 isolates and a selection of 12 recombinant HIV-1 clones. Except for two dual-tropic (CCR5/CXCR4) clinical isolates, all of the clones tested use the CCR5 coreceptor for entry. We compared the antiviral activity of the new generation of inhibitors with that of the previously described inhibitor Ref2,¹⁰ which has structural similarity to 8. 33 exhibited anti-HIV-1 activity similar to that of Ref2, as demonstrated by their overall mean IC₅₀ values and their antiviral activities against the different HIV subtypes (Table 2). The overall mean IC₅₀ value for Ref2 was 0.39 ± 0.02 μM (IC₅₀ values ranged from 0.19 to 0.79 μM), whereas the overall mean IC₅₀ value determined for 33 was 0.43 ± 0.02 μM (IC₅₀ values ranged from 0.13 to 0.99 μM). Ref2 showed better antiviral activities against subtype D viruses (mean IC₅₀ value of 0.26 ± 0.03 μM), whereas 33 showed better antiviral activities against subtype C and D viruses (mean IC₅₀ values of 0.37 ± 0.03 and 0.35 ± 0.07 μM, respectively). In contrast, the respective SI values (CC₅₀/IC₅₀) determined for the panel of pseudoviruses were higher for 33 than for Ref2, with 1.5–2.1-fold improvements, depending on the viral subtype. Furthermore, this new generation of compounds exhibited a better cytotoxicity profile (8: CC₅₀ of 109.3 ± 2 μM and 33: CC₅₀ of 85 ± 3 μM) than that for Ref2 (CC₅₀ of 42.4 ± 1.0 μM). 8 showed better SI values than both Ref2 and 33, as well as better anti-HIV-1 activities and a better cytotoxicity profile. The overall mean IC₅₀ value for 8 was 0.18 ± 0.008 μM (IC₅₀ values ranged from 0.11 to 0.33 μM). Except for subtype A (mean IC₅₀ value of 0.22 ± 0.003 μM), 8 worked equally well against all of the subtypes tested (mean IC₅₀ value of 0.17–0.18 μM). The SI value calculated using the overall mean IC₅₀ value was 607.2, and depending on the viral subtype, the SI values varied from 497 to 643, representing 4.9–5.6-fold improvements compared with Ref2. Overall, 8 and 33 were active against all of the tested clinical isolates, regardless of the viral subtypes (A–D), indicating that these compounds have broad-spectrum inhibitory activity, unlike BMS-378806, an early stage attachment inhibitor, which despite its low nanomolar potency showed weaker activity against A, C, and D subtypes.³³ Furthermore, 8 and 33, similar to earlier reports for Ref2, were poorly active against the pseudovirus VSV-G, which was used here as a control, suggesting that the inhibitory activities of these compounds are specific to HIV-1. Additionally, 8 and 33 did not induce toxicity in the U87-CD4-CXCR4 cell line at the doses used for this assay.

Table 2.

Neutralization Activity of gp120 Entry Antagonists against a Panel of HIV-1 Env Pseudoviruses

				IC50,a μM
subtype	NIH No.	ENVs	Ref2	33	8
A	11887	Q259ENV.W6	0.46 ± 0.06b	0.4 ± 0.05	0.23 ± 0.03
	11888	QB726.70M.ENV.C4	0.31 ± 0.07	0.63 ± 0.1	0.22 ± 0.01
	11890	QF495.23M.ENV.A1	0.44 ± 0.05	0.47 ± 0.05	0.14 ± 0.003
	11891	QF495.23M.ENV.A3	0.26 ± 0.04b	0.71 ± 0.17	0.33 ± 0.01
		BG505-T332N	0.41 ± 0.03	0.27 ± 0.007	0.124 ± 0.005
		KNH1144	0.62 ± 0.08	0.74 ± 0.06	0.26 ± 0.02
A/D	11901	QA790.204I.ENV.A4	0.29 ± 0.01	0.36 ± 0.03	0.14 ± 0.003
	11904	QA790.204I.ENV.E2	0.41 ± 0.03	0.36 ± 0.02	0.16 ± 0.03
A2/D	11905	QG393.60M.ENV.A1	0.34 ± 0.02	0.42 ± 0.03	0.22 ± 0.002
	11906	QG393.60M.ENV.B7	0.6 ± 0.005	0.5 ± 0.04	0.17 ± 0.03
A/G	11591	CRF02_AG Clone 211	0.58 ± 0.05	0.38 ± 0.04	0.23 ± 0.04
	11594	CRF02_AG clone 250	0.41 ± 0.01	0.42 ± 0.01	0.21 ± 0.01
	11595	CRF02_AG clone 251	0.36 ± 0.06	0.39 ± 0.03	0.12 ± 0.01
	11598	CRF02_AG clone 255	0.33 ± 0.01	0.66 ± 0.03	0.22 ± 0.01
	11599	CRF02_AG clone 257	0.39 ± 0.01	0.74 ± 0.1	0.16 ± 0.002
	11600	CRF13 cpx clone 258	0.52 ± 0.07	0.52 ± 0.1	0.14 ± 0.003
	11602	CRF02_AG clone 266	0.51 ± 0.06	0.75 ± 0.05	0.23 ± 0.01
AE	11603	CRF01_AE clone 269	0.52 ± 0.02	0.41 ± 0.2	0.138 ± 0.01
B		B41	0.36 ± 0.04	0.38 ± 0.05	0.136 ± 0.001
	11018	QH0692, clone 42	0.27 ± 0.01b	0.99 ± 0.05	0.28 ± 0.02
	11022	PVO, clone 4	0.41 ± 0.06	0.33 ± 0.04	0.11 ± 0.03
	11023	TRO, clone 11	0.39 ± 0.02	0.45 ± 0.03	0.23 ± 0.005
	11036	RHPA4259 clone 7	0.37 ± 0.07	0.24 ± 0.09	0.15 ± 0.01
	11037	THRO4156 clone 18	0.32 ± 0.02	0.15 ± 0.03	0.16 ± 0.02
	11038	CAAN5342 clone A2	0.22 ± 0.03	0.38 ± 0.07	0.13 ± 0.005
	11058	SC422661.8	0.32 ± 0.08	0.16 ± 0.01	0.13 ± 0.01
	11560	1006_11.C3.1601	0.58 ± 0.06	0.27 ± 0.01	0.139 ± 0.003
	11561	1054.TC4.1499	0.25 ± 0.02	0.39 ± 0.02	0.23 ± 0.01
	11562	1056.TA11.1826	0.58 ± 0.01	0.71 ± 0.1	0.13 ± 0.006
	11563	1058 11.B11.1550c	0.29 ± 0.02	0.49 ± 0.06	0.28 ± 0.01
	11572	9021_14.B2.4571	0.36 ± 0.08	0.57 ± 0.13	0.11 ± 0.03
	11578	WEAUd15.410.5017c	0.76 ± 0.04	0.54 ± 0.04	0.22 ± 0.002
C	11307	Du172, clone 17	0.3 ± 0.03	0.27 ± 0.02	0.19 ± 0.002
	11308	Du422, clone 1	0.63 ± 0.09	0.42 ± 0.01	0.23 ± 0.01
	11309	ZM197M.PB7, SVPC6	0.27 ± 0.01	0.31 ± 0.03	0.13 ± 0.006
	11310	ZM214M.PL15, SVPC7	0.24 ± 0.007	0.26 ± 0.005	0.13 ± 0.005
	11312	ZM249M.PL1, SVPC10	0.55 ± 0.07	0.44 ± 0.1	0.18 ± 0.01
	11313	ZM53M.PB12, SVPC11	0.54 ± 0.03	0.57 ± 0.3	0.28 ± 0.002
	11314	ZM109F.PB4	0.29 ± 0.01	0.47 ± 0.01	0.28 ± 0.003
	11317	CAP210.2.00.E8, SVPC17	0.47 ± 0.03	0.14 ± 0.05	0.115 ± 0.002
	11502	HIV-16055–2, clone 3	0.29 ± 0.06	0.29 ± 0.09	0.12 ± 0.006
	11504	HIV-16936–2, clone 21	0.47 ± 0.05	0.48 ± 0.1	0.21 ± 0.04
	11506	HIV-25711–2, clone 4	0.26 ± 0.02	0.32 ± 0.03	0.17 ± 0.01
	11507	HIV-225925–2, clone 22	0.24 ± 0.02	0.46 ± 0.04	0.17 ± 0.01
	11908	QB099.391M.ENV.B1	0.42 ± 0.05	0.33 ± 0.05	0.118 ± 0.002
D	11911	QA013.70I.ENV.H1	0.32 ± 0.01	0.55 ± 0.04	0.25 ± 0.01
	11912	QA013.70I.ENV.M12	0.22 ± 0.01	0.25 ± 0.02	0.16 ± 0.02
	11916	QD435.100M.ENV.B5	0.3 ± 0.01	0.37 ± 0.03	0.13 ± 0.01
	11918	QD435.100M.ENV.E1	0.19 ± 0.04	0.24 ± 0.01	0.136 ± 0.002
G	11596	CRF02_G clone 252	0.26 ± 0.03	0.13 ± 0.01	0.11 ± 0.01
mean ± SEM (μM): overall (n = 50) SI			0.39 ± 0.02 108.7	0.43 ± 0.02 198.4	0.18 ± 0.008 607.2
subtype A (n = 6) SI			0.42 ± 0.05 101	0.54 ± 0.08 158	0.22 ± 0.003 496.8
subtype Arec (n = 12) SI			0.44 ± 0.03 96.4	0.49 ± 0.04 174.1	0.18 ± 0.01 607.2
subtype B (n = 14) SI			0.39 ± 0.04 108.7	0.43 ± 0.06 198.4	0.17 ± 0.02 642.9
subtype C (n = 13) SI			0.38 ± 0.04 111.6	0.37 ± 0.03 230.5	0.18 ± 0.02 607.2
subtype D (n = 4) SI			0.26 ± 0.03 163.1	0.35 ± 0.07 243.7	0.17 ± 0.03 642.9
control	VSV-Gd	IC50	>20	>20	>20
		CC50	46.3 ± 10.6	>94	>94
IC50 (color code)			≥0.2	>0.2 < 0.5	>0.5

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

^bData previously published.¹⁰

^cR5 × 4-tropic virus; all the rest are CCR5-tropic viruses.

^dVSV-G was tested in U87-CD4-CCR5 cells.

Moreover, we tested the anti-HIV-1 activity of 8 and 33 against an HIV-1 panel, comprised of paired infant and maternal clones belonging to subtypes A and D/A. These HIV clones were isolated from chronically infected mothers and their respective infected infants.³⁴ Previous studies have shown that vertically transmitted HIV infant variants were more difficult to neutralize using combinations of broadly neutralizing antibodies (bNAbs) 2G12, biz, 2F5, and 4E10.³⁴ In this study, we found that Ref2, 8, and 33 equally neutralized both the infant and maternal HIV-1 variants (Table 3), as shown by their overall mean IC₅₀ values and the means for infant and maternal viruses. 8, the most effective compound with an overall mean IC₅₀ value of 0.37 ± 0.07 μM, was slightly more efficient against the maternal clones (mean IC₅₀ value of 0.31 ± 0.04 μM) than against the infant clones (mean IC₅₀ value of 0.43 ± 0.07 μM). These findings suggest that the new gp120 entry antagonists can neutralize both infant and maternal HIV-1 variants.

Table 3.

Neutralization Activity of gp120 Entry Antagonists against an HIV-1 Panel of Paired Infant (B) and Maternal (M) Env Molecular Clones

			IC50a (μM)
subtype	NIH No.	ENV	Ref2	33	8
A	11518-B	BG505.W6M.ENV.C2	0.92 ± 0.16	1.38 ± 0.2	0.57 ± 0.06
A	11528-M	MG505.W0M.ENV.A2	0.93 ± 0.07	1.6 ± 0.08	0.47 ± 0.08.
A	11519-B	B1206.W6P.ENVA1A	0.75 ± 0.06	1.8 ± 0.07	0.53 ± 0.05
A	11531-M	MI206.W0M.ENV.D1	0.7 ± 0.02	1 ± 0.02	0.28 ± 0.01
A	11521-B	BJ613.W6M.ENV.E1	0.8 ± 0.07	0.94 ± 0.14	0.26 ± 0.01
A	11535-M	MJ613.W0M.ENV.A2	0.5 ± 0.05	0.63 ± 0.07	0.31 ± 0.07
D/A	11524-B	BL035.W6M.ENV.C1	0.52 ± 0.16	0.54 ± 0.04	0.42 ± 0.01
D/A	11538-M	ML035.W0M.ENV.G2	0.59 ± 0.08	0.86 ± 0.05	0.23 ± 0.01
A	11525-B	BL274.W6M.ENV.A3	0.63 ± 0.12	0.84 ± 0.09	0.37 ± 0.05
A	11540-M	ML274.W0M.ENV.B1	0.66 ± 0.1	0.88 ± 0.06	0.25 ± 0.03
mean ± SEM (μM): overall (n = 10)			0.7 ± 0.05	1.05 ± 0.13	0.37 ± 0.04
infant (B) (n = 5)			0.72 ± 0.07	1.1 ± 0.22	0.43 ± 0.07
mother (M) (n = 5)			0.68 ± 0.07	0.99 ± 0.16	0.31 ± 0.04

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

Inhibitory Activity of 8 and 33 against a Large Panel of FDA-Approved Drug-Resistant Viruses.

To further evaluate the neutralizing activities of 8 and 33, we assessed these compounds against a large set of drug-resistant viruses, including five Enfuvirtide (T-20)-resistant viruses, seven multidrug, non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant viruses, which carry mutations that confer resistance to both NNRTIs and nucleoside inhibitors (NRTIs), three Raltegravir-resistant viruses, and nine protease inhibitor (PI)-resistant viruses (Table 4). As a reference, the activities of the gp120 entry antagonists were assessed against the reconstructed wild-type (WT) HIV-1_NL4–3 clone, from which the drug-resistant clones were obtained. For this assay, the WT HIV-1_NL4–3 control virus or the drug-resistant viruses were pretreated with the gp120 entry antagonists and then used to infect TZMb-l cells. We found that 8 and 33 both inhibited WT HIV-1_NL4–3, with IC₅₀ values of 2.1 and 1.1 μM, respectively. Moreover, both compounds neutralized the T-20-resistant viruses, with IC₅₀ values similar to the IC₅₀ values determined for the control virus, WT HIV-1_NL4–3. Similar results were observed for the NNRTI-resistant viruses, and in some cases, the drug-resistant viruses appeared to be more sensitive to the gp120 entry antagonists than the WT virus. The Raltegravir-resistant viruses and the PI-resistant viruses were also highly sensitive to 8 and 33, as shown by their low IC₅₀ values. For 33, the IC₅₀ values detected against these clones were 1.8–10.5-fold lower than the IC₅₀ value detected for WT HIV-1_NL4–3, and for 8, the IC₅₀ values were 1.8–11.7-fold lower than the IC₅₀ value detected for WT HIV-1_NL4–3. In conclusion, we found that 8 and 33 were active against all of the tested drug-resistant viruses, indicating that these compounds could potentially be successfully used in combination with other antiviral agents.

Table 4.

Inhibitory Activity of 8 and 33 against a Large Panel of FDA Approved Drug-Resistant Viruses

	NIH catalog No.	major mutationsa	33 IC50b (μM)	fold increase/sensitive	8 IC50b (μM)	fold increase/sensitive
NL4–3 WT (wild-type)	114		2.1 ± 0.1		1.1 ± 0.04
ENTRY (Enfavirtide)-resistant	9498	V38A, N42T	1.5 ± 0.2	sensitive	0.94 ± 0.1	sensitive
	9490	V38A	1.9 ± 0.1	sensitive	0.94 ± 0.02	sensitive
	9496	V38E, N42S	1.6 ± 0.2	sensitive	0.89 ± 0.1	sensitive
	9491	N42T, N43K	2.5 ± 0.2	sensitive	0.47 ± 0.02	sensitive
	9489	D36G	0.73 ± 0.1	sensitive	0.71 ± 0.01	sensitive
multidrug (NNRTI) resistant	12227	K101P, K103N	0.48 ± 0.01	sensitive	0.23 ± 0.01	sensitive
	12229	L100I, K103N	2.9 ± 0.1	1.38	2.4 ± 0.2	2.18
	12231	K103N, Y181C	0.42 ± 0.04	sensitive	0.26 ± 0.02	sensitive
	12233	K101E, Y181 V	3.7 ± 0.3	1.76	2.4 ± 0.07	2.18
	12237	Y181C, G190A	3.5 ± 0.5	1.66	2 ± 0.1	1.82
	12241	K101E, G190S	1.7 ± 0.3	sensitive	0.47 ± 0.01	sensitive
	12243	L100I, M230L	4.5 ± 0.05	2.14	2.3 ± 0.05	2.09
integrase (Raltegravir-resistant)	11847	G140S, Q148H	0.2 ± 0.04	sensitive	0.23 ± 0.005	sensitive
	11850	E92Q, N155H	0.72 ± 0.22	sensitive	0.3 ± 0.03	sensitive
	11851	N155H	0.26 ± 0.1	sensitive	0.094 ± 0.005	sensitive
multiple-PI-resistant	11800	11I, 32I, 33F, 46I, 47 V, 54M, 58E, 73S, 84 V, 89 V, 90M	0.28 ± 0.05	sensitive	0.16 ± 0.07	sensitive
	11801	10F, 33F, 43T, 46L, 54 V, 82A, 84 V, 90M	0.31 ± 0.16	sensitive	0.19 ± 0.1	sensitive
	11803	33F, 43T, 46I, 48 V, 50 V, 54S, 82A	1.2 ± 0.2	sensitive	0.61 ± 0.2	sensitive
	11804	32I, 46I, 47 V, 84 V	0.51 ± 0.07	sensitive	0.26 ± 0.04	sensitive
	11805	48 V, 53L, 54 V, 82A, 90M	1 ± 0.1	sensitive	0.27 ± 0.14	sensitive
	11807	32I, 33F, 47A, 82A, 90M	0.58 ± 0.1	sensitive	0.12 ± 0.02	sensitive
	11808	10F, 11I, 33F, 43T, 46L, 54 V, 73S, 82A, 84 V, 89 V, 90M	0.47 ± 0.1	sensitive	0.73 ± 0.1	sensitive
	12465	46I, 54 V, 58E, 74P, 82L, 90M	1 ± 0.2	sensitive	0.42 ± 0.1	sensitive
	12466	32I, 33F, 43T, 46I, 47 V, 54M, 73S, 82A, 89 V, 90M	0.47 ± 0.1	sensitive	0.24 ± 0.09	sensitive

^aMutants were reported here as per the data obtained from https://www.aidsreagent.org/ and associated references indicated in the Experimental Section.

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

However, we are currently conducting experiments to identify the possible generation of drug-resistant mutants in response to the use of the most potent gp120 entry antagonists examined in this study.

gp120 Entry Antagonists Inhibited Cell-to-Cell HIV-1 Transmission.

Another important feature described for the earlier generation of gp120 entry antagonists was their ability to inhibit cell-to-cell HIV-1 transmission,¹⁰ which has been reported to be more efficient than HIV-1 cell-free infection.^35,36 Multiple viral particles can be transmitted to noninfected cells simultaneously. Additionally, cell-to-cell HIV-1 transmission in vitro has been shown to be resistant to some potent bNAbs, including CD4 binding site (CD4bs) antibodies^36–38 and NRTIs, but not to other antiretrovirals, including entry inhibitors, NNRTIs, and protease inhibitors.³⁹

Here, we evaluated the activities of the new generation of gp120 entry antagonists, 8 and 33, against cell-to-cell HIV-1 transmission and compared their activities to that of Ref2, which has previously been reported to inhibit cell-to-cell HIV-1 transmission.¹⁰ H9 cells, chronically infected with HIV-1_IIIB (CXCR4-tropic), and MOLT-4 cells, chronically infected with HIV-1_ADA (CCR5-tropic), were used as donor cells, and TZM-bl cells were used as acceptor cells. BMS-626529 was used as a control treatment drug. Our results (Table 5) indicated that both 8 and 33 have similar activities to that for the previous generation compound, Ref2, against cell-to-cell HIV transmission. All of the tested compounds, including BMS-626529, showed better activities against the CXCR4-tropic virus HIV-1_IIIB than against the CCR5-tropic virus HIV-1_ADA. In particular, the IC₅₀ values for the gp120 entry antagonists calculated against HIV-1_IIIB in the CXCR4-tropic assay were onefold lower than the IC₅₀ values calculated against HIV-1_ADA in the CCR5-tropic assay.

Table 5.

Inhibitory Activity against Cell-to-Cell HIV Transmission by the gp120 Entry Antagonists

	IC50a (μM)
compound	TZMb-l/H9-HIV-1IIIB	TZMb-l/Molt-HIV-1ADA
Ref2	0.46 ± 0.2	0.89 ± 0.14
33	0.45 ± 0.1	1.2 ± 0.4
8	0.47 ± 0.1	0.92 ± 0.13
BMS-626529	0.02 ± 0.005	~0.2

^aThe reported IC₅₀ values represent the means ± standard deviation (SD), n = 3.

In Vitro ADME Assessment.

The in vitro assessment of ADME properties has played an important role, especially for the pharmaceutical industry, in reducing the drug attrition rate. In 1997, the major causes of failure for drugs that advanced to clinical trials were poor ADME properties.⁴⁰ However, the judicious use of ADME assessments during the early stages of drug development has dramatically improved the failure rate of drugs during recent years.⁴¹ Drug failures during the later stages of drug development can be very costly. Therefore, the major goal of the pharmaceutical industry with regard to drug development programs is to “fail early, fail cheaply”.⁴¹ Therefore, we also adopted in vitro ADME assessments in our early optimization phase of developing these entry antagonists as future clinical candidates. We previously reported the ADME properties of our most active inhibitor, Ref1, which targets the Phe43 cavity of gp120, and compared our results with the data obtained for BMS-626529, a prodrug of which is showing promising results in Phase III clinical trials.¹⁰ The ADME properties of Ref1 indicated room for further improvements.

Since this is the first time we decided to use a pyridine scaffold as an aromatic ring in Region I and this bioisostere was used before in medicinal chemistry to improve physicochemical and ADME properties, it was also imperative for us to evaluate these properties by selecting one of our most active inhibitors, 8, which has a better cytotoxicity profile and SI values than the other compounds examined in this study and compared with Ref1 previously reported.¹⁰ The data presented in Table 6 shows that 8 (a pyridine analogue) has approximately threefold improved solubility compared with Ref1 (a phenyl analogue) and BMS-626529, which was expected and was the basis of the design strategy used in this study. Since these inhibitors are expected to be used orally, we performed the Caco-2 bidirectional permeability experiment [apical to basolateral (A-B) and basolateral to apical (B-A) across the Caco-2 cell monolayer], which can be used to measure the efflux ratio and predict the human intestinal permeability of orally administered drugs. The data shown in Table 6 indicates that the apparent permeabilities of 8 and the clinical candidate, BMS-626529, are similar. However, the permeability of Ref1 was poor compared with both of these inhibitors. We used digoxin, a P-gp substrate, as a positive control to identify whether active efflux was mediated by P-gp. It is apparent that the efflux of 8, Ref1, and digoxin was mediated by P-gp. However, we were unable to directly compare the effects of P-gp inhibitors on permeability parameters, because different P-gp inhibitors were used in the current study (1 μM valsopodar) and the previous study (100 μM verapamil).¹⁰ However, the data confirmed that all three compounds had efflux ratios greater than 2, suggesting the potential involvement of an efflux transporter, which can mediate the transport of these inhibitors from the basolateral side to the apical side. Interestingly, the efflux ratios for all three cases were reduced in the presence of a P-gp inhibitor. For 8, BMS-626529, and digoxin (positive control) the efflux ratios were reduced to less than 2, indicating that these compounds could be P-gp substrates. However, for Ref1, the efflux ratio was not reduced to less than 2, indicating the possible involvement of other transporters, such as breast cancer-resistant protein (BCRP) or multidrug resistance-associated protein 2 (MRP2).

Table 6.

In Vitro ADME Profile of the Most Potent Inhibitor 8

		compound
assay performed	in vitro ADMET	8	Ref1a	BMS-626529a	positive control used in the study (digoxin)
solubility (mg/mL)	phosphate buffer, pH7.4	0.734	0.042–0.214	0.047–0.237
Caco-2 permeability (mean Papp × 10−6 cm/sec)	A-to-B	6.51	0.602	9.27	0.483
	B-to-A	20.3	17.7	32.0	10.3
	efflux ratio	3.12	30.5	3.46	21.2
(+1 μM Valspodar)	A-to-B	14.0	0.777b	13.5b	2.00
	B-to-A	10.5	10.9b	22.7b	2.06
	efflux ratio	0.755	14.4b	1.69b	1.03
metabolic stability (human liver microsomes)	parent compound remaining at 120 min (% of 0 min)	93.5	88.5	71.5
	Clint (mL/min/mg protein)	<0.0116	0.0018	0.0052
	Half-life (min)	>120
protein binding (human plasma)	% bound	99.2	99.0	86.9
cytochrome P450 inhibition, IC50 (μM)	CYP1A2 (Phenacetin)	70.1	>25	>25
	CYP2B6 (Bupropion)	85.4	>25	>25
	CYP2C8 (Amodiaquine)	>100	>25c	>25c
	CYP2C9 (Diclofenac)	>100	>25	>25
	CYP2C19 (S-Mephenytoin)	>100	>25	>25
	CYP2D6 (Bufuralol)	>100	>25	>25
	CYP3A (Testosterone)	>62.2	>25	>25
	CYP3A (Midazolam)	>100	>25	>25

^aThe data were from ref 10.

^b100 μM verapamil was used as a P-gp inhibitor

^cPaclitaxel was used as a substrate.

We also examined the metabolic stability of 8 in the human liver microsome, because the liver is the primary site of drug metabolism. The data in Table 6 shows that 8 has the highest stability (93.5%) among the examined compounds, followed by Ref1, whereas BMS-626529 was metabolized to some degree (71.5%) in our earlier study. The clearance data (Cl_int) indicated that all three compounds are low-clearance compounds, and 8 had a half-life longer than 120 min. We did not calculate half-life data in our earlier reported study. It is worthwhile to mention that compounds with high clearance values may not be considered favorable, because they may be cleared rapidly from the body, and the drugs may have a short duration of action may need multiple dosing. We also examined the protein-binding potential of these inhibitors in human plasma. The data indicated that both 8 and Ref1 were highly bound (>99%), whereas BMS-626529 had a binding potential of 86.9%.

The oxidative biotransformation of many lipophilic drugs into hydrophilic counterparts is critical, facilitating the elimination of drugs from the body. The cytochrome P450 enzyme system plays a critical role in these oxidative biotransformation reactions associated with drug metabolism. More than 50 CYP450 enzymes have been identified, but CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 metabolize almost 80% of all drugs. Therefore, we decided to use this set of eight CYP450 enzymes to determine whether any of our potent inhibitors have inhibitory effects that may help predict potential drug–drug interactions when coadministered with other treatment agents. Earlier, we reported the inhibitory effects of Ref1 and BMS-626529, which showed no inhibitory activities for doses as high as 25 μM. In this study, we escalated the tested doses to as high as 100 μM, and our most potent inhibitor, 8, showed no inhibition at greater than 50 μM dose levels against CYP1A2, CYP2B6, and CYP3A. No inhibitory activities were observed against the other CYP450 enzymes at the 100 μM dose level.

CONCLUSIONS

Here, we reported a significant shift in the design of gp120 antagonists, based on our previously reported and optimized inhibitor, Ref1. The phenyl ring in this class of inhibitors has been accepted to represent the critical moiety for antiviral potency, because this ring is located deep inside the narrow hydrophobic cavity, according to X-ray crystallography data. In this study, we deployed pyridine, a bioisostere of phenyl, as a scaffold to replace the phenyl ring. We synthesized 48 novel compounds and determined a comprehensive SAR to identify the best possible gp120 entry antagonists that display improved cytotoxicity and ADME properties. We identified the two best inhibitors, 8 and 33, as determined by antiviral activity against HIV-1_HXB2. These inhibitors were confirmed to be gp120 entry antagonists and showed broad-spectrum activity against a large panel of HIV-1 Env-pseudotyped viruses, representing diverse subtypes. Although the antiviral potency of these two new inhibitors remained similar to that for Ref1, the cytotoxicity of 8 was substantially improved, with an overall SI value of 607 against 50 diverse clinical isolates. This improvement was quite substantial compared to the SI value for 33, which was 198. The ADME data for 8 also showed a considerable improvement in aqueous solubility. All other ADME properties for 8 were comparable to those for the clinical candidate BMS-626529, a prodrug of which is currently being tested in Phase III clinical trials. Overall, the pyridine substitution of the phenyl ring has been the most effective alteration in our quest to identify a clinically relevant gp120 antagonist. This finding is expected to improve the antiviral potency, cytotoxicity, and ADME properties of this class of inhibitors for subsequent preclinical studies.

EXPERIMENTAL SECTION

Cells and Viruses.

TZM-bl cells,⁴² U87CD4+CXCR4+ cells,⁴³ HIV-1 IIIB, infected H9 cells,⁴⁴ and MOLT-4 CCR5+ cells⁴⁵ were obtained through the National Institutes of Health AIDS Reagent Program (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.⁴⁶ HIV-1 Env molecular clone expression vector pHXB2-env (X4) DNA was also obtained through the NIH ARP.⁴⁷ HIV-1 Env molecular clones of gp160 genes for HIV-1 Env pseudovirus production were obtained as follows: clones representing the standard panels A, A/D, A2/D, D, and C (QB099.391M.Env.B1) were obtained through the NIH ARP from Dr. J. Overbaugh.^48,49 The HIV-1 Env molecular clones panel of subtype A/G, A/E, and G Env clones were obtained through the NIH ARP from Drs. D. Ellenberger, B. Li, M. Callahan, and S. Butera.⁵⁰ The HIV-1 Env panel of standard reference subtype B Env clones was obtained through the NIH ARP from Drs. D. Montefiori, F. Gao, and M. Li (PVO, clone 4 (SVPB11) TRO, clone 11 (SVPB12), QH0692, clone 42 (SVPB6), SC422661, clone B (SVPB8)); from Drs. B. H. Hahn and J. F. Salazar-Gonzalez (pRHPA4259, clone 7 (SVPB14)); from Drs. B. H. Hahn and D. L. Kothe (pTHRO4156 clone 18 (SVPB15), pCAAN5342 clone A2 (SVPB19)).^51,52 The subtype B clones pWEAUd15.410.5017, p1058_11.B11.1550, p1054.TC4.1499, p1006_11.C3.1601, p1056.TA11.1826, and p9021_14.B2.4571 were obtained through the NIH ARP from Drs. B. H. Hahn, B. F. Keele, and G. M. Shaw.⁵³ The subtype C HIV-1 reference panel of Env clones was also obtained through the NIH ARP from Drs. D. Montefiori, F. Gao, S. A. Karim, and G. Ramjee (Du172.17); from Drs. D. Montefiori, F. Gao, C. Williamson, and S. A. Karim (Du422.1), from Drs. B. H. Hahn, Y. Li and J. F. Salazar-Gonzalez (ZM197M.PB7; ZM214M.PL15, ZM249M.PL1); from Drs. E. Hunter and C. Derdeyn (ZM53M.PB12; ZM109F.PB4); from Drs. L. Morris, K. Mlisana, and D. Montefiori, (CAP210.2.00.E8).^54–56 The HIV-1 subtype C panel of Indian gp160 Env clones HIV-16055–2 clone 3, HIV-16936–2 clone 21, HIV-25711–2 clone 4 and HIV-225925–2 clone 22 were obtained through the NIH ARP from Drs. R. Paranjape, S. Kulkarni, and D. Montefiori.⁵⁰ The panel of paired infant and maternal HIV-1 Env molecular clones were obtained through the NIH ARP from Dr. J. Overbaugh.⁵⁷ The Env pseudotyped genes of BG505.T332N, KNH1144, and B41 were kindly provided by Dr. J. P. Moore of the Weil Cornell Medical College.

The Env-deleted proviral backbone plasmids pNL4–3.Luc.R-.E-DNA (from Dr. N. Landau),^58,59 the pSG3Δ^env DNA (from Drs. J. C. Kappes and X. Wu),^42,52 and the pNL4–3 (from Dr. Malcolm Martin)⁶⁰ were obtained through the NIH ARP Division of AIDS, NIAID, NIH. The following molecular clones were also obtained through the NIH ARP, Division of AIDS, NIAID, NIH: the panel of Enfuvirtide (T-20) resistant viruses from Trimeris, Inc.;^60,61 the panel multidrug resistant NNRTI infectious clones from Dr. R. Shafer;⁶² the Raltegravir-resistant infectious molecular clones from Dr. R. Shafer and E. Reuman, M.S.,⁶³ and the multidrug protease inhibitor-resistant infectious clones from Dr. R. Shafer.⁶⁴

MLV gag-pol-expressing vector pVPack-GP, Env-expressing vector pVPack-VSV-G, and a pFB-Luc vector were obtained from Stratagene.

Pseudovirus Preparation.

Pseudoviruses capable of single-cycle infection were prepared as previously described.^27,31 Briefly, 5 × 10⁶ HEK293T cells were transfected with an HIV-1 Env-deleted pro-viral backbone plasmid pSG3^Δenv or pNL4–3.Luc.R-.E-DNA, and an HIV-1 Env-expression plasmid by using FuGENE6 (Promega). The control VSV-G pseudovirus was prepared by transfecting the HEK293T cells with a combination of the Env-expressing plasmid pVPack-VSV-G, the MLV gag-pol-expressing plasmid pVPack-GP, the pFB-Luc plasmid, and FuGENE6. Pseudovirus-containing supernatantswere collected 2 d after transfection, filtered, titered, and stored in aliquots at −80 °C.

Measurement of Antiviral Activity.

Single-Cycle Infection Assay in TZM-bl Cells.

The antiviral activity of the gp120 entry antagonists was evaluated in single-cycle infection assay by infecting TZM-bl cells with HIV-1 pseudotyped with the Env from the lab-adapted HIV-1_HXB‑2 (CXCR4-tropic). Additionally, Ref2, 33, and 8 were tested against a large group of HIV-1 pseudotyped with the Env from the panel of clinical isolates as previously described.^27,31 Briefly, TZM-bl cells were plated at 1 × 10⁴/well in a 96-well tissue culture plate and cultured overnight. On the following day, HIV-1 pseudovirus was pretreated with graded concentrations of the small molecules for 30 min and added to the cells. After 3 d of incubation, the cells were washed and lysed with 50 μL of lysis buffer (Promega). Twenty microliters of the lysates were transferred to a white plate and mixed with the luciferase assay reagent (Promega). The luciferase activity was measured immediately with a Tecan Spark reader, and the percent inhibition by the compounds and the half-maximal inhibitory concentration values were calculated using the GraphPad Prism software.

Single-Cycle Infection Assay in U87-CD4-CXCR4 Cells.

The antiviral activity of Ref2, 33, and 8 was tested against the control pseudovirus VSV-G in U87-CD4-CXCR4 cells. Briefly, U87-CD4-CXCR4 cells were plated in a 96-well tissue culture plate at 1 × 10⁴/well and cultured at 37 °C. The following day, aliquots of pseudovirus pretreated with graded concentrations of the small molecules for 30 min were added to the cells and incubated for 3 d. 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.

Assay in Cf2Th-CCR5 Cells.

CD4-negative Cf2Th-CCR5 cells were plated at 6 × 10³ cells/well in a 96-well tissue culture plate and incubated at 37 °C. The cells were infected with the recombinant CD4-dependent pseudovirus HIV-1_ADA as previously described.¹⁸ Briefly, aliquots of HIV-1_ADA pseudovirus pretreated with graded concentrations of compounds for 30 min were added to the cells and cultured for 48 h. Cells were washed with phosphate-buffered solution (PBS) and lysed with 40 μL of cell lysis reagent. Lysates were transferred to a white 96-well plate and mixed with the luciferase assay reagent. The luciferase activity was immediately measured to obtain the relative infection compared to the untreated control. The relative virus infectivity indicates the ratio of the amount of infection detected in the presence of the compounds and the amount of infection detected in the absence of the compounds.

Measurement of Antiviral Activity against Drug-Resistant Viruses in TZM-bl Cells.

The antiviral activity of the gp120 entry antagonists against a panel of drug-resistant viruses was evaluated by infecting TZM-bl cells. Briefly, TZM-bl cells were plated at 10⁴/well in a 96-well plate and cultured overnight. On the following day, HIV-1 drug-resistant viruses were pretreated with graded concentrations of the small molecules for 30 min and added to the cells. Following 48 h of incubation, the cells were washed and lysed. The cellular lysates were transferred to a white plate, and the luciferase activity was immediately measured with a Tecan Spark reader. The percent inhibition by the compounds and the IC₅₀ values were calculated using the GraphPad Prism software, as reported above.

Cell-to-Cell HIV-1 Transmission.

The cell-to-cell HIV-1 transmission inhibition assay was performed as previously described,^{65, 66} with a few modifications. Briefly, TZM-bl cells (used as acceptor cells) wereplated at 10⁴/well in a 96-well plate 24 h before the assay. As transmitting cells, we used H9 cells chronically infected with HIV-1_IIIB at 2 × 10³ cells/well for the CXCR4-tropic assay and MOLT-4/CCR5 cells chronically infected with HIV-1_ADA at 2 × 10³ cells/well for the CCR5-tropic assay. The transmitting cells were pretreated with 200 μg/mL mitomycin C (Sigma) for 1 h at 37 °C, washed with PBS, and incubated with the acceptor cells in the presence of escalating concentrations of compounds for 24 h. Therefore, the cells were washed and lysed. The lysates were 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.

Evaluation of Cytotoxicity.

TZM-bl Cells and U87-CD4-CXCR4 Cells.

The cytotoxicity of the gp120 entry antagonists in TZM-bl and U87-CD4-CXCR4 cells was measured by using the colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) (Promega) following the manufacturer’s instructions. Briefly, the cells were plated at 1 × 10⁴/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 d. The MTS reagent was added to the cells and incubated for 4 h at 37 °C. The absorbance was recorded at 490 nm. The percent of cytotoxicity and the the concentration for 50% cytotoxicity values were calculated as above.

Cf2Th-CCR5 Cells.

The cytotoxicity of the small molecules in Cf2Th-CCR5 cells was also measured with the colorimetric CellTiter 96 AQueous One Solution Cell Proliferation Assay. Briefly, Cf2Th-CCR5 cells were plated in a 96-well plate and cultured at 37 °C overnight. Next, the cells were incubated with 100 μL of the compounds at graded concentrations and cultured for 48 h. The MTS reagent was added to the cells, and 4 h later, the absorbance was recorded at 490 nm. The percent of cytotoxicity and the CC₅₀ values were calculated as above.

In Vitro ADME Study.

Details of the in vitro ADME study and data analyses can be found in the Supplemental Information.

Chemistry.

General.

We used commercial reagents and solvents without further purification. We also performed all reactions in the air atmosphere unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) performed on Merck TLC Silica gel plates (60 F254), using a UV light for visualization and basic aqueous potassium permanganate or iodine fumes as a developing agent. ¹H and ¹³C NMR spectra were recorded on Bruker Avance 400 instrument with operating frequency of 400 and 100 MHz, respectively, and calibrated using residual undeuterated chloroform (δH = 7.28 ppm) and CDCl₃ (δC = 77.16 ppm) or undeuterated dimethyl sulfoxide (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, m = multiplet, br = broad. The purity of the final compounds was checked by liquid chromatography-mass spectrometry (LCMS) in a Shimadzu LCMS-2010A using three types of detection systems such as EDAD, ELSD, and UV and was found to be at least 95%.

General Procedure A: For Suzuki Coupling.

To a solution containing appropriate bromide (or chloride) (50 mmol, 1 equiv), (1-(tert-butoxycarbonyl)-1H-pyrrol-2-yl)boronic acid (50 mmol, 1 equiv) in tetrahydrofuran (THF)–H₂O (1:1, 100 mL), Na₂CO₃ (100 mmol, 2 equiv) and Pd(Ph₃P)Cl₂ (1 mol %) were added under a nitrogen atmosphere. The mixture was stirred at reflux for 8–15 h (TLC-control). After it cooled to room temperature, water (50 mL) and CH₂Cl₂ (50 mL) were added. The organic layer was separated; the aqueous layer was extracted with CH₂Cl₂ (3 × 50 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated. Purification by flash chromatography using the hexane (Hex)–ethyl acetate (EtOAc) mixture as eluent afforded the desired compound. Compounds S1a–S1h were obtained following the general procedure A. Compounds S1g and S1h were obtained from the corresponding chlorides.

tert-Butyl 2-(5-meth ylpyridin-2-yl)-1H-pyrrole-1-carboxylate (S1a).

Eluent: Hex–EtOAc (from 10:1 to 5:1), R_f = 0.2 (5:1, Hex-EtOAc). Yield = 63%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.37 (s, 9 H), 2.34 (s, 3 H), 6.22 (t, J = 3.3 Hz, 1 H), 6.36 (dd, J = 3.2, 1.7 Hz, 1 H), 7.28 (d, J = 8.1 Hz, 1 H), 7.33 (dd, J = 3.2, 1.8 Hz, 1 H), 7.48 (dd, J = 7.9, 1.8 Hz, 1 H), 8.42–8.45 (m, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 18.3, 27.6 (3C), 83.5, 110.5, 115.3, 123.2, 123.2, 131.3, 134.2, 136.3, 149.2, 149.3, 150.2.

tert-Butyl 2-(5-chloropyridin-2-yl)-1H-pyrrole-1-carboxylate (S1b).

Eluent: Hex–EtOAc (from 20:1 to 10:1), R_f = 0.3 (10:1, Hex-EtOAc). Yield = 84%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.41 (s, 9 H), 6.25 (t, J = 3.3 Hz, 1 H), 6.43 (dd, J = 3.3, 1.7 Hz, 1 H), 7.34–7.38 (m, 2 H), 7.66 (dd, J = 8.4, 2.5 Hz, 1 H), 8.57 (d, J = 2.4 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 27.8 (3C), 84.1, 110.8, 116.3, 124.0, 124.3, 130.2, 133.1, 135.6, 147.8, 149.2, 151.0

tert-Butyl 2-(5-fluoropyridin-2-yl)-1H-pyrrole-1-carboxylate (S1c).

Eluent: Hex-EtOAc (from 20:1 to 10:1), R_f = 0.3 (10:1, Hex-EtOAc). Yield = 68%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.39 (s, 9 H), 6.23 (t, J = 3.3 Hz, 1 H), 6.39 (dd, J = 3.3, 1.7 Hz, 1 H), 7.35 (dd, J = 3.2, 1.7 Hz, 1 H), 7.39 (d, J = 1.8 Hz, 1 H), 7.40–7.42 (m, 1 H), 8.47 (t, J = 1.7 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 27.7 (3C), 83.9, 110.6, 115.8, 122.7 (d, J = 18.6 Hz), 123.6, 124.6 (d, J = 4.2 Hz), 133.1, 137.0 (d, J = 23.8 Hz), 149.2 (d, J = 1.5 Hz), 149.3, 158.4 (d, J = 256.0 Hz).

tert-Butyl 2-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-1-carboxylate (S1d).

Eluent: Hex-EtOAc (from 30:1 to 20:1), R_f = 0.3 (30:1, Hex-EtOAc). Yield = 65%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.42 (s, 9 H), 6.28 (t, J = 3.3 Hz, 1 H), 6.54 (dd, J = 3.4, 1.7 Hz, 1 H), 7.40 (dd, J = 3.2, 1.7 Hz, 1 H), 7.54 (d, J = 8.2 Hz, 1 H), 7.92 (dd, J = 8.3, 2.2 Hz, 1 H), 8.87 (s, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 27.6 (3C), 84.3, 110.9, 117.3, 122.8, 123.8 (q, J = 272.0 Hz), 124.4 (q, J = 33.0 Hz), 124.8, 132.9 (q, J = 3.5 Hz), 133.0, 145.8 (q, J = 4.1 Hz), 149.1, 156.0 (q, J = 1.5 Hz).

tert-Butyl 2-(6-chloropyridin-3-yl)-1H-pyrrole-1-carboxylate (S1e).

Eluent: Hex-EtOAc (from 20:1 to 10:1), R_f = 0.4 (10:1, Hex-EtOAc). Yield = 76%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.44 (s, 9 H), 6.25–6.29 (m, 2 H), 7.33 (d, J = 8.2 Hz, 1 H), 7.41 (t, J = 2.5 Hz, 1 H), 7.66 (dd, J = 8.2, 2.5 Hz, 1 H), 8.38 (d, J = 2.3 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 27.8 (3C), 84.6, 111.1, 116.1, 123.1, 123.8, 129.3, 130.0, 139.4, 149.0, 149.5, 150.0.

tert-Butyl 2-(6-(trifluoromethyl)pyridin-3-yl)-1H-pyrrole-1-carboxylate (S 1f).

Eluent: Hex-EtOAc (from 30:1 to 20:1), R_f = 0.5 (30:1, Hex-EtOAc). Yield = 84%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.43 (s, 9 H), 6.30 (t, J = 3.3 Hz, 1 H), 6.32–6.35 (m, 1 H), 7.45 (dd, J = 3.2, 1.8 Hz, 1 H), 7.68 (d, J = 8.1 Hz, 1 H), 7.86 (dd, J = 8.1, 1.8 Hz, 1 H), 8.72 (d, J = 1.7 Hz, 1 H).

¹³C NMR (CDCl₃, 400 MHz).

δ = 27.7 (3C), 84.8, 111.3, 116.8, 119.5 (q, J = 2.8 Hz), 121.8 (q, J = 273.9 Hz), 124.3, 130.0, 133.2, 137.3, 146.3 (q, J = 34.8 Hz), 148.9, 150.0.

tert-Butyl 2-(6-(trifluoromethyl)pyridazin-3-yl)-1H-pyrrole-1-carboxylate (S1g).

Eluent: Hex-EtOAc (from 20:1 to 5:1), R_f = 0.4 (5:1, Hex-EtOAc). Yield = 61%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.45 (s, 9 H), 6.35 (t, J = 3.4 Hz, 1 H), 6.73 (dd, J = 3.4, 1.6 Hz, 1 H), 7.48 (dd, J = 3.2, 1.7 Hz, 1 H), 7.75–7.79 (m, 2 H).

¹³C NMR (CDCl₃, 400 MHz).

δ = 27.8 (3C), 85.1, 111.6, 119.2, 121.7 (q, J = 274.2 Hz), 122.8 (q, J = 2.4 Hz), 125.8, 127.7, 129.9, 148.9, 149.5 (q, J = 35.0 Hz), 157.2.

tert-Butyl 2-(5-chloropyrimidin-2-yl)-1H-pyrrole-1-carboxylate (S1h).

Eluent: Hex-EtOAc (from 30:1 to 20:1), R_f = 0.5 (30:1, Hex-EtOAc). Yield = 58%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.44 (s, 9 H), 6.26 (t, J = 3.3 Hz, 1 H), 6.78 (dd, J = 3.4, 1.7 Hz, 1 H), 7.35 (dd, J = 3.1, 1.7 Hz, 1 H), 8.67 (s, 2 H).

¹³C NMR (CDCl₃, 400 MHz).

δ = 27.8 (3C), 84.3, 110.9, 118.8, 125.7, 128.4, 131.8, 149.2, 155.2 (2C), 158.8.

General Procedure B: for Boc-deprotection.

To a solution containing Boc-protected compound (30 mmol) in MeOH (15 mL, 2 M solution), 1 M HCl solution in MeOH (45 mL) was added in one portion. The mixture was stirred at reflux for 7–8 h. After this mixture cooled to room temperature, the solvent was evaporated. Then 10% aqueous K₂CO₃ (50 mL) was added carefully (CO₂ evolution), and the mixture was extracted with CH₂Cl₂ (3 × 50 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated. Crude product was used in the next step without purification. Compounds S2a–S2h were obtained following the general procedure B.

5-Methyl-2-(1H-pyrrol-2-yl)pyridine (S2a).

Yield = 94%.

¹H NMR (CDCl₃, 400 MHz).

δ = 2.33 (s, 3 H), 6.31–6.35 (m, 1 H), 6.72–6.75 (m, 1 H), 6.89–6.91 (m, 1 H), 7.48 (dd, J = 8.2, 1.9 Hz, 1 H), 7.53 (d, J = 8.1 Hz, 1 H), 8.27–8.38 (m, 1 H), 10.73 (br. s., 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 18.2, 106.8, 109.9, 118.1, 119.9, 129.9, 131.8, 137.4, 148.5, 148.9.

5-Chloro-2-(1H-pyrrol-2-yl)pyridine (S2b).

Yield = 92%.

¹H NMR (CDCl₃, 400 MHz).

δ = 6.31–6.34 (m, 1 H), 6.71–6.74 (m, 1 H), 6.92 (td, J = 2.6, 1.4 Hz, 1 H), 7.50 (d, J = 8.6 Hz, 1 H), 7.60 (dd, J = 8.6, 2.4 Hz, 1 H), 8.42 (d, J = 2.2 Hz, 1 H), 9.90 (br. s., 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 107.9, 110.6, 118.9, 120.5, 128.3, 130.7, 136.4, 147.7, 148.9.

5-Fluoro-2-(1H-pyrrol-2-yl)pyridine (S2c).

Yield = 93%.

¹H NMR (CDCl₃, 400 MHz).

δ = 6.33 (dd, J = 6.2, 2.7 Hz, 1 H), 6.66–6.71 (m, 1 H), 6.88–6.94 (m, 1 H), 7.38 (td, J = 8.5, 2.8 Hz, 1 H), 7.56 (dd, J = 8.8, 4.3 Hz, 1 H), 8.35 (d, J = 2.8 Hz, 1 H), 9.96 (br. s., 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 107.1, 110.3, 119.1 (d, J = 4.1 Hz), 120.2, 123.9 (d, J = 19.3 Hz), 130.9, 136.7 (d, J = 23.9 Hz), 147.4 (d, J = 3.2 Hz), 157.8 (d, J = 253.2 Hz).

2-(1H-Pyrrol-2-yl)-5-(trifluoromethyl)pyridine (S 2d).

Eluent: Hex-EtOAc (from 20:1 to 10:1), R_f = 0.5 (10:1, Hex-EtOAc). Yield = 88%.

¹H NMR (CDCl₃, 400 MHz).

δ = 6.32–6.37 (m, 1 H), 6.80–6.86 (m, 1 H), 6.94–7.03 (m, 1 H), 7.61 (d, J = 8.4 Hz, 1 H), 7.83 (dd, J = 8.4, 2.1 Hz, 1 H), 8.71 (s, 1 H), 9.74 (br. s., 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 109.6, 111.0, 117.6, 121.6 122.9 (q, J = 33.0 Hz), 124.0 (q, J = 271.6 Hz), 130.5, 133.7 (q, J = 3.5 Hz), 146.1 (q, J = 4.4 Hz), 153.5 (q, J = 1.5 Hz).

2-Chloro-5-(1H-pyrrol-2-yl)pyridine (S2e).

Yield = 93%.

¹H NMR (CDCl₃, 400 MHz).

δ = 6.33–6.37 (m, 1 H), 6.57–6.60 (m, 1 H), 6.94–6.98 (m, 1 H), 7.32 (d, J = 8.4 Hz, 1 H), 7.74 (dd, J = 8.3, 2.6 Hz, 1 H), 8.52 (d, J = 2.4 Hz, 1 H), 8.84 (br. s., 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 107.9, 110.6, 120.8, 124.5, 127.4, 128.2, 134.2, 144.6.

5-(1H-Pyrrol-2-yl)-2-(trifluoromethyl)pyridine (S 2f).

Yield = 92%.

¹H NMR (CDCl₃, 400 MHz).

δ = 6.29–6.41 (m, 1 H), 6.64–6.74 (m, 1 H), 6.92–7.04 (m, 1 H), 7.63 (d, J = 8.2 Hz, 1 H), 7.89 (dd, J = 8.2, 1.8 Hz, 1 H), 8.82 (d, J = 1.8 Hz, 1 H), 9.32 (br. s., 1 H).

¹³C NMR (CDCl₃, 400 MHz).

δ = 109.2, 111.0, 120.9 (q, J = 2.9 Hz), 121.8, 121.8 (q, J = 273.7 Hz), 127.2, 131.5, 131.7, 144.7 (q, J = 35.1 Hz), 144.9.

3-(1H-Pyrrol-2-yl)-6-(trifluoromethyl)pyridazine (S2g).

Yield = 95%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 6.25–6.29 (m, 1 H), 7.07–7.14 (m, 2 H), 8.10 (d, J = 9.0 Hz, 1 H), 8.20 (d, J = 9.0 Hz, 1 H), 12.15 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 110.5, 112.7, 122.0 (q, J = 273.5 Hz), 122.9, 124.2, 124.8 (q, J = 2.2 Hz), 126.9, 147.3 (q, J = 33.7 Hz), 154.8.

5-Chloro-2-(1H-pyrrol-2-yl)pyrimidine (S2h).

Yield = 90%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 6.20 (s, 1 H), 6.93 (d, J = 1.8 Hz, 1 H), 6.97 (s, 1 H), 8.78 (s, 2 H), 11.77 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 110.1, 112.3, 123.2, 125.7, 129.4, 155.6 (2C), 157.1.

General Procedure C: For Acylation.

Crude pyrrole from the previous step (1 equiv) was dissolved in CH₂Cl₂ (0.5 M solution), and pyridine (1.2 equiv) was added, followed by dropwise addition of trifluoroacetic anhydride (TFAA) (1.2 equiv). After completion of the addition, the mixture was stirred for 1 h, and the solvent was evaporated. The product was triturated in aqueous, and the precipitate was filtered, washed with water twice, and dried on a filter. Compounds S3a–S3h were obtained following the general procedure C.

2,2,2-Trifluoro-1-(5-(5-methylpyridin-2-yl)-1H-pyrrol-2-yl)-ethanone (S3a).

Yield = 89%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 2.34 (s, 3 H), 7.05 (d, J = 4.2 Hz, 1 H), 7.28 (dd, J = 3.9, 1.9 Hz, 1 H), 7.75 (dd, J = 8.0, 1.4 Hz, 1 H), 8.09 (d, J = 8.1 Hz, 1 H), 8.49–8.52 (m, 1 H), 12.87 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 17.8, 111.8, 117.0 (q, J = 290.3 Hz), 120.6, 122.9 (q, J = 3.5 Hz), 126.1, 133.4, 137.8, 142.6, 145.3, 149.8, 168.1 (q, J = 35.0 Hz).

1-(5-(5-Chloropyridin-2-yl)-1H-pyrrol-2-yl)-2,2,2-trifluoroethanone (S3b).

Yield = 94%.

¹H NMR (DMSO-d₆, 400 MHz).

δ =7.09 (dd, 1 H), 7.29 (dt, J = 4.1, 2.0 Hz, 1 H), 8.07 (dd, J = 8.6, 2.5 Hz, 1 H), 8.23 (d, J = 8.6 Hz, 1 H), 8.69 (d, J = 2.4 Hz, 1 H), 13.04 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 112.5, 117.0 (q, J = 290.25 Hz), 122.0, 122.7 (q, J = 3.5 Hz), 126.6, 130.7, 137.0, 141.5, 146.7, 148.3, 168.5 (q, J = 35.0 Hz).

2,2,2-Trifluoro-1-(5-(5-fluoropyridin-2-yl)-1H-pyrrol-2-yl)-ethanone (S3c).

Yield = 91%.

¹H NMR (DMSO-d₆, 400 MHz).

δ =7.04 (dd, J = 4.1, 2.3 Hz, 1 H), 7.28 (dt, J = 4.1, 2.0 Hz, 1 H), 7.87 (td, J = 8.8, 2.9 Hz, 1 H), 8.27 (dd, J = 8.9, 4.4 Hz, 1 H), 8.65 (d, J = 2.8 Hz, 1 H), 12.96 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 112.0, 117.0 (q, J = 289.9 Hz), 122.4 (d, J = 4.8 Hz), 122.7 (q, J = 3.5 Hz), 124.1 (d, J = 19.0 Hz), 126.4, 138.0 (d, J = 24.5 Hz), 141.8, 144.9 (d, J = 3.9 Hz), 158.7 (d, J = 256.2 Hz), 168.3 (q, J = 35.0 Hz).

2,2,2-Trifluoro-1-(5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrol-2-yl)ethanone (S 3d).

Yield = 92%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 7.19 (dd, J = 4.0, 2.2 Hz, 1 H), 7.27–7.32 (m, 1 H), 8.33 (dd, J = 8.4, 1.7 Hz, 1 H), 8.40 (d, J = 8.3 Hz, 1 H), 8.99 (s, 1 H), 13.19 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 113.7, 117.2 (q, J = 289.8 Hz), 120.9, 122.7 (q, J = 1.5 Hz), 124.0 (q, J = 271.5 Hz), 124.5 (q, J = 32.2 Hz), 127.5, 135.1, 141.2, 146.7, 152.0, 169.1 (q, J = 34.4 Hz).

1-(5-(6-Chloropyridin-3-yl)-1H-pyrrol-2-yl)-2,2,2-trifluoroethanone (S3e).

Yield = 87%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 7.08 (dd, J = 4.2, 2.4 Hz, 1 H), 7.33 (dt, J = 4.1, 2.0 Hz, 1 H), 7.64 (d, J = 8.4 Hz, 1 H), 8.43 (dd, J = 8.4, 2.6 Hz, 1 H), 9.02 (d, J = 2.4 Hz, 1 H), 13.14 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 111.9, 117.1 (q, J = 289.9 Hz), 123.2 (q, J = 3.3 Hz), 124.5, 125.5, 126.8, 137.0, 139.1, 147.6, 150.2, 168.3 (q, J = 35.0 Hz).

2,2,2-Trifluoro-1-(5-(6-(trifluoromethyl)pyridin-3-yl)-1H-pyrrol-2-yl)ethanone (S 3f).

Yield = 93%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 7.14 (d, J = 4.3 Hz, 1 H), 7.26–7.33 (m, 1 H), 7.96 (d, J = 8.3 Hz, 1 H), 8.61 (dd, J = 8.2, 1.8 Hz, 1 H), 9.32 (d, J = 1.7 Hz, 1 H), 13.24 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 112.6, 116.9 (q, J = 289.8 Hz), 120.8 (q, J = 2.2 Hz), 121.6 (q, J = 273.7 Hz), 122.9 (q, J = 3.7 Hz), 127.2, 129.1, 135.1, 138.4, 145.7 (q, J = 34.4 Hz), 147.7, 168.5 (q, J = 35.1 Hz).

2,2,2-Trifluoro-1-(5-(6-(trifluoromethyl)pyridazin-3-yl)-1H-pyrrol-2-yl)ethanone (S3g).

Yield = 85%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 7.34–7.39 (m, 2 H), 8.39 (d, J = 9.0 Hz, 1 H), 8.70 (d, J = 9.0 Hz, 1 H), 13.50 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 114.1, 116.7 (q, J = 289.8 Hz), 121.6 (q, J = 274.0 Hz), 122.3 (q, J = 3.1 Hz), 125.4 (q, J = 2.0 Hz), 125.5, 128.0, 137.8, 149.2 (q, J = 34.1 Hz), 153.8, 169.1 (q, J = 35.4 Hz).

1-(5-(5-Chloropyrimidin-2-yl)-1H-pyrrol-2-yl)-2,2,2-trifluoroethanone (S3h).

Yield = 86%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 7.10 (d, J = 3.9 Hz, 1 H), 7.24 (s, 1 H), 8.97 (s, 2 H), 13.07 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 114.2, 116.7 (q, J = 289.8 Hz), 122.2 (q, J = 2.9 Hz), 127.2, 129.2, 139.6, 155.3, 156.2 (2C), 168.7 (q, J = 35.1 Hz).

General Procedure D: For Haloform Reaction.

Appropriate trifluoroethanone (1 equiv) was added to a solution of NaOH (5 equiv) in dioxane–H₂O mixture (1:1, 0.5 M solution). The resulting reaction mixture was refluxed for 20 h and cooled to room temperature. A concentrated aqueous HCl solution (~12 M, 5 equal) was added dropwise. The resulting precipitate was filtered off, washed with H₂O, and dried on a filter. Compounds S4a–S4h were obtained following general procedure D.

5-(5-Methylpyridin-2-yl)-1H-pyrrole-2-carboxylic Acid (S4a).

Yield = 82%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 2.28 (s, 3 H), 6.77–6.84 (m, 2 H), 7.61 (dd, J = 8.1, 2.1 Hz, 1 H), 7.88 (d, J = 8.1 Hz, 1 H), 8.38 (d, J = 1.8 Hz, 1 H), 11.69 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 17.9, 109.2, 116.5, 119.1, 124.8, 131.6, 136.4, 137.6, 147.1, 149.7, 162.0.

5-(5-Chloropyridin-2-yl)-1H-pyrrole-2-carboxylic Acid (S4b).

Yield = 73%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 6.78–6.93 (m, 2 H), 7.94 (dd, J = 8.6, 2.4 Hz, 1 H), 8.06 (d, J = 8.6 Hz, 1 H), 8.58 (d, J = 2.2 Hz, 1 H), 11.96 (br. s., 1 H), 12.57 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 110.3, 116.2, 120.3, 125.8, 128.8, 135.0, 136.8, 147.9, 148.2, 161.8.

5-(5-Fluoropyridin-2-yl)-1H-pyrrole-2-carboxylic Acid (S4c).

Yield = 85%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 6.80–6.85 (m, 2 H), 7.74 (td, J = 8.8, 2.9 Hz, 1 H), 8.08 (dd, J = 8.8, 4.3 Hz, 1 H), 8.53 (d, J = 2.8 Hz, 1 H), 11.86 (br. s., 1 H), 12.52 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 109.7, 116.3, 120.6 (d, J = 4.4 Hz), 124.1 (d, J = 18.8 Hz), 125.1, 135.4, 137.4 (d, J = 24.0 Hz), 146.4 (d, J = 3.7 Hz), 158.0 (d, J = 253.4 Hz), 161.8.

5-(5-(Trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxylic Acid S 4d).

Yield = 80%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 6.76–6.95 (m, 1 H), 6.95–7.08 (m, 1 H), 8.12–8.31 (m, 2 H), 8.89 (s, 1 H), 12.18 (br. s., 1 H), 12.71 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 111.7, 116.3, 119.0, 122.7 (q, J = 32.2 Hz), 124.0 (q, J = 272.2 Hz), 126.7, 134.4 (q, J = 3.7 Hz), 134.8, 146.2 (q, J = 4.4 Hz), 153.1, 161.8.

5-(6-Chloropyridin-3-yl)-1H-pyrrole-2-carboxylic acid (S4e).

Yield = 91%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 6.73–6.89 (m, 2 H), 7.52 (d, J = 8.4 Hz, 1 H), 8.29 (dd, J = 8.4, 2.6 Hz, 1 H), 8.90 (d, J = 2.4 Hz, 1 H), 12.26 (br. s., 1 H), 12.51 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 109.2, 116.4, 124.2, 125.5, 127.1, 132.0, 135.7, 146.3, 148.2, 161.8.

5-(6-(Trifluoromethyl)pyridin-3-yl)-1H-pyrrole-2-carboxylic Acid (S 4f).

Yield = 80%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 6.84–6.87 (m, 1 H), 6.87–6.90 (m, 1 H), 7.85 (d, J = 8.3 Hz, 1 H), 8.45 (dd, J = 8.3, 1.5 Hz, 1 H), 9.20 (d, J = 1.2 Hz, 1 H), 12.40 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 110.5, 116.6, 120.9 (q, J = 2.9 Hz), 243.9 (q, J = 273.7 Hz), 126.5, 130.8, 131.8, 133.5, 144.2 (q, J = 33.7 Hz), 146.7, 161.9.

5-(6-(Trifluoromethyl)pyridazin-3-yl)-1H-pyrrole-2-carboxylic Acid (S4g).

Yield = 87%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 6.91 (dd, J = 3.9, 2.3 Hz, 1 H), 7.20 (dd, J = 3.9, 2.4 Hz, 1 H), 8.27 (d, J = 9.0 Hz, 1 H), 8.57 (d, J = 9.0 Hz, 1 H), 12.55 (br. s., 1 H), 12.83 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 112.7, 116.4, 121.9 (q, J = 273.7 Hz), 123.9, 125.1 (q, J = 2.2 Hz), 127.9, 131.7, 148.2 (q, J = 33.7 Hz), 154.7, 161.6.

5-(5-Chloropyrimidin-2-yl)-1H-pyrrole-2-carboxylic Acid (S4h).

Yield = 75%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 6.81 (d, J = 3.7 Hz, 1 H), 6.96 (d, J = 3.7 Hz, 1 H), 8.87 (s, 2 H), 11.63 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 112.7, 115.7, 127.4, 128.1, 132.9, 155.9 (2C), 156.2, 161.7.

tert-Butyl 2-(2-fluoropyridin-4-yl)-1H-pyrrole-1-carboxylate (S5).

Compound S5 was obtained following the general procedure A. Yield = 67%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.45 (s, 9 H), 6.24–6.27 (m, 1 H), 6.36 (dd, J = 3.3, 1.7 Hz, 1 H), 6.88–6.91 (m, 1 H), 7.14–7.17 (m, 1 H), 7.41 (dd, J = 3.2, 1.7 Hz, 1 H), 8.16 (d, J = 5.2 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 27.7, 84.9, 109.0 (d, J = 38.3 Hz), 111.2, 117.0, 121.6 (d, J = 3.9 Hz), 124.8, 131.2 (d, J = 3.9 Hz), 146.6 (d, J = 15.5 Hz), 147.1 (d, J = 8.9 Hz), 148.8, 163.6 (d, J = 237.2 Hz).

2-Methoxy-4-(1H-pyrrol-2-yl)pyridine (S6).

Compound S6 was obtained following the general procedure B. Yield 92%.

¹H NMR (CDCl₃, 400 MHz).

δ = 3.96 (s, 3 H), 6.31–6.36 (m, 1 H), 6.68–6.74 (m, 1 H), 6.78 (d, J = 1.2 Hz, 1 H), 6.93 (s, 1 H), 6.99 (dd, J = 5.5, 1.5 Hz, 1 H), 8.11 (d, J = 5.5 Hz, 1 H), 8.89 (br. s., 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 53.7, 103.8, 109.0, 110.7, 112.3, 121.0, 129.3, 142.4, 147.3, 165.1.

2,2,2-Trifluoro-1-(5-(2-methoxypyridin-4-yl)-1H-pyrrol-2-yl)-ethanone (S7).

Compound S7 was obtained following the general procedure C. Yield = 78%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 3.88 (s, 3 H), 7.10 (dd, J = 4.2, 2.4 Hz, 1 H), 7.28 (dt, J = 4.0, 1.9 Hz, 1 H), 7.46 (s, 1 H), 7.54 (dd, J = 5.4, 1.4 Hz, 1 H), 8.21 (d, J = 5.4 Hz, 1 H), 13.12 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 53.4, 106.5, 112.4, 114.0, 116.9 (q, J = 290.1 Hz), 122.8 (q, J = 3.5 Hz), 126.8, 139.6, 140.2, 147.7, 164.5, 168.5 (q, J = 35.0 Hz).

5-(2-Methoxypyridin-4-yl)-1H-pyrrole-2-carboxylic acid (S8).

Compound S8 was obtained following the general procedure D. Yield = 93%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 3.86 (s, 3 H), 6.80–6.84 (m, 1 H), 6.84–6.88 (m, 1 H), 7.35 (s, 1 H), 7.43 (dd, J = 5.5, 1.4 Hz, 1 H), 8.11 (d, J = 5.5 Hz, 1 H), 12.26 (br. s., 1 H), 12.56 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 53.2, 104.9, 110.2, 113.2, 116.2, 125.9, 133.5, 141.3, 147.2, 161.7, 164.5.

Synthesis of Reagent Methyl 2-((tert-butoxycarbonyl)-amino)-3-iodobut-2-enoate (R4).

Methyl 2-((tert-butoxycarbonyl)amino)but-2-enoate (R3).

4-Dimethylaminopyridine (DMAP) (3.64 g, 0.1 equiv) was added to a solution of the N-Boc acid methyl ester R1 (69.59 g, 1 equiv) in dry acetonitrile (300 mL), followed by di-tert-butyl dicarbonate (65.0 g, 1.0 equiv) with rapid stirring at room temperature. The reaction was monitored by TLC (diethyl ether/n-hexane, 1:1), until all the reactants had been consumed. Tetramethylguanidine (TMG) (3.44 mL, 0.1 equiv) was then added, stirring was continued, and the reaction was followed by TLC. When all the reactants had been consumed, evaporation at reduced pressure gave a residue that was partitioned between CH₂Cl₂ (300 mL) and 5% HCl (200 mL). The organic phase was thoroughly washed with NaHCO₃ and dried with Na₂SO₄. Removal of the solvent afforded the corresponding N-Boc-dehydroamino acid methyl ester. Mass (M) = 63.88 g.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.48 (s, 9 H), 1.82 (d, J = 7.2 Hz, 3 H), 3.78 (s, 3 H), 6.00 (br. s, 1 H), 6.69 (q, J = 7.0 Hz, 1 H).

Methyl 2-((tert-butoxycarbonyl)amino)-3-iodobut-2-enoate (R4).

A flask was charged with the dehydroamino acid derivative R3 (63.88 g, 48.4 mmol), K₂CO₃ (81.7 g, 2 equiv), and THF (400 mL). I₂ (90.9 g, 1.2 equiv) was added, and the reaction mixture was heated at reflux for ~4 h. After the system had cooled to room temperature, the reaction mixture was quenched with a 10% solution of Na₂SO₃ (100 mL). The mixture was extracted with CH₂Cl₂ (3 × 100 mL). The combined organic layers were dried over Na₂SO₄, filtered, and evaporated. The residue was subjected to column chromatography (eluent hexanes/EtOAc, 10:1). M = 52.2 g.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.47 (s, 9 H), 2.74 (s, 3 H), 3.83 (s, 3 H), 6.17 (br. s, 1 H).

5-(Trifluoromethyl)-2-((trimethylsilyl)ethynyl)pyridine (S9).

In a pressurized vessel equipped with magnetic stirring bar containing solution of 2-bromo-5-(trifluoromethyl)pyridine (30 g; 133 mmol, 1 equiv) in Et₃N (265 mL), TMS–acetylene (26.07 g, 36.8 mL, 265 mmol, 2 equiv), Pd(Ph₃P)Cl₂ (0.93 g; 1 mol %), and CuI (0.51 g; 2 mol %) were added under argon atmosphere. It was heated to 50–60 °C, and the mixture was stirred at this temperature for 6–8 h (TLC-control). Water (500 mL) was added and extracted with hexane (3 × 150 mL). The combined extracts were washed with water and dried with anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the residue was purified using column chromatography (eluent: Hex–EtOAc, 30:1, R_f = 0.5 in hexane-EtOAc 30:1). M = 15.6 g. Yield = 48%.

¹H NMR (CDCl₃, 400 MHz).

δ = 0.28 (s, 9 H), 7.55 (d, J = 8.2 Hz, 1 H), 7.87 (dd, J = 8.2, 2.2 Hz, 1 H), 8.81 (s, 1 H).

¹³C NMR (CDCl₃, 400 MHz).

δ = −0.4 (3C), 98.2, 102.5, 123.3 (q, J = 272.2 Hz), 125.6 (q, J = 33.3 Hz), 126.9, 133.4 (q, J = 3.1 Hz), 146.5, 146.8 (q, J = 3.8 Hz).

5-Chloro-2-((trimethylsilyl)ethynyl)pyridine (S10).

In a pressurized vessel equipped with magnetic stirring bar containing a solution of 2-bromo-5-chloropyridine (20 g; 104 mmol, 1 equiv) in Et₃N (210 mL), TMS-acetylene (20.42 g, 28.8 mL, 208 mmol, 2 equiv), Pd(Ph₃P)Cl₂ (0.73 g; 1 mol %), and CuI (0.4 g; 2 mol %) were added under argon atmosphere. It was heated to 50–60 °C, and the mixture was stirred at this temperature for 6–8 h (TLC-control). Water (500 mL) was added and extracted with hexane (3 × 150 mL). The combined extracts were washed with water and dried with anhydrous sodium sulfate. The solvent was removed by rotary evaporation, and the residue was purified using column chromatography (eluent: Hex-EtOAc, 30:1, R_f = 0.4 in hexane-EtOAc 30:1). M = 16.7 g. Yield = 74%.

¹H NMR (CDCl₃, 400 MHz).

δ = 0.27 (s, 9 H), 7.40 (d, J = 8.4 Hz, 1 H), 7.62 (dd, J = 8.4, 2.4 Hz, 1 H), 8.52 (d, J = 2.4 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = −0.3 (3c), 96.1, 102.6, 127.8, 131.5, 135.9, 141.0, 148.9.

1-tert-Butyl 2-methyl 3-methyl-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-1,2-dicarboxylate (S11).

To the solution of S9 (9.81 g, 40 mmol, 1 equiv) in dimethylformamide (DMF) (200 mL), triethylamine trihydrofluoride (1.98 g, 2 mL, 12 mmol, 0.3 equiv) was added, and the mixture was stirred for 1 h under argon atmosphere. Then methyl (E)-2-(tert-butoxycarbonylamino)-3-iodo-but-2-enoate (13.75 g, 1 equiv), Pd(Ph₃P)Cl₂ (1.42 g; 5 mol %), CuI (0.77 g; 10 mol %), and Cs₂CO₃ (26.27 g; 80 mmol, 2 equiv) were added under argon atmosphere. It was heated to 70–80 °C, and the mixture was stirred at this temperature for 10–15 h (TLC-control). Water (300 mL) was added and extracted with Et₂O (3 × 150 mL). Combined extracts were washed with water and brine, dried over Na₂SO₄, filtered, and concentrated. Purification by flash chromatography was done using hexane-EtOAc mixture (20:1) as eluent. M = 7.1 g, Yield 46%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.64 (s, 9 H), 2.34 (s, 3 H), 3.89 (s, 3 H), 6.52 (s, 1 H), 7.64 (d, J = 8.1 Hz, 1 H), 7.89 (d, J = 7.8 Hz, 1 H), 8.73 (br. s., 1 H).

¹³C NMR (CDCl₃, 400 MHz).

δ = 13.1, 27.4 (3C), 51.5, 85.1, 114.1, 120.5, 123.1, 123.6 (q, J = 272.2 Hz), 124.3 (q, J = 32.9 Hz),129.2, 133.2, 133.6 (q, J = 2.9 Hz), 145.1 (q, J = 4.4 Hz), 149.9, 152.5, 161.2.

1-tert-Butyl 2-methyl 5-(5-chloropyridin-2-yl)-3-methyl-1H-pyrrole-1,2-dicarboxylate (S12).

To the solution of S10 (13.1 g, 62 mmol, 1 equiv) in DMF (310 mL), triethylamine trihydrofluoride (3.01 g, 3.05 mL, 12 mmol, 0.3 equiv) was added, and mixture was stirred for 1 h under argon atmosphere. Then methyl (E)-2-(tert-butoxycarbonylamino)-3-iodo-but-2-enoate (21.31 g, 1 equiv), Pd(Ph₃P)Cl₂ (1.32 g; 3 mol %), CuI (1.2 g; 10 mol %), and Cs₂CO₃ (40.7 g; 125 mmol, 2 equiv) were added under argon atmosphere. It was heated to 70–80 °C, and the mixture was stirred at this temperature for 10–15 h (TLC-control). Water (300 mL) was added and extracted with Et₂O (3 × 150 mL). Combined extracts were washed with water and brine, dried over Na₂SO₄, filtered, and concentrated. M = 8.95 g, Yield 41%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.61 (s, 9 H), 2.33 (s, 3 H), 3.87 (s, 3 H), 6.41 (s, 1 H), 7.49 (d, J = 8.5 Hz, 1 H), 7.65 (dd, J = 8.5, 2.4 Hz, 1 H), 8.45 (d, J = 2.1 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 13.3, 27.5 (3C), 51.6, 85.1, 113.2, 122.0, 122.4, 129.5, 130.5, 133.9, 136.3, 147.3, 147.8, 150.1, 161.4.

Methyl 3-methyl-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxylate (S13).

To a solution of S11 (5.87 g, 15 mmol, 1 equiv) in CH₂Cl₂ (50 mL) trifluoroacetic acid (TFA) (11 g, 7.4 mL, 76 mmol, 5 equiv) was added in one portion, and the mixture was stirred overnight. Then solvent was evaporated; 10% aqueous K₂CO₃ was added, and the mixture was extracted with CH₂Cl₂ (3 × 100 mL), dried over Na₂SO₄, filtered, and concentrated. Purification by flash chromatography was done using hexane-EtOAc mixture (10:1) as eluent. M = 3.96 g. Yield 91%.

¹H NMR (CDCl₃, 400 MHz).

δ = 2.40 (s, 3 H), 3.90 (s, 3 H), 6.62 (d, J = 2.8 Hz, 1 H), 7.61 (d, J = 8.3 Hz, 1 H), 7.87 (dd, J = 8.4, 2.1 Hz, 1 H), 8.76 (s, 1 H), 10.01 (br. s., 1 H).

¹³C NMR (CDCl₃, 400 MHz).

δ = 12.8, 51.4, 112.1, 118.4, 121.5, 123.7 (q, J = 272.2 Hz), 124.1 (q, J = 32.9 Hz), 129.6, 132.4, 133.8 (q, J = 3.7 Hz), 146.4 (q, J = 4.4 Hz), 152.1, 161.6.

Methyl 5-(5-chloropyridin-2-yl)-3-methyl-1H-pyrrole-2-carboxylate (S14).

To a solution of S12 (8.25 g, 24 mmol, 1 equiv) in CH₂Cl₂ (100 mL) TFA (13.4 g, 9.0 mL, 118 mmol, 5 equiv) was added in one portion, and the mixture was stirred overnight. Then solvent was evaporated; 10% aqueous K₂CO₃ was added, and the mixture was extracted with CH₂Cl₂ (3 × 100 mL), dried over Na₂SO₄, filtered, and concentrated. Purification by flash chromatography was done using hexane-EtOAc mixture (10:1) as eluent. M = 5.76 g. Yield 98%.

¹H NMR (CDCl₃, 400 MHz).

δ = 2.38 (s, 3 H), 3.88 (s, 3 H), 6.50 (d, J = 2.6 Hz, 1 H), 7.46 (d, J = 8.4 Hz, 1 H), 7.62 (dd, J = 8.5, 2.4 Hz, 1 H), 8.45 (d, J = 2.2 Hz, 1 H), 9.89 (br. s., 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 12.9, 51.4, 110.9, 119.8, 120.7, 129.7, 129.9, 132.7, 136.5, 147.4, 148.2, 161.7.

Sodium 3-methyl-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxylate (S15).

To a solution of S13 (3.96 g, 14 mmol, 1 equiv) in a mixture of dioxane–H₂O (1:1, 30 mL), NaOH (0.61 g, 15 mmol, 1.1 equiv) was added in one portion, and the reaction mixture was stirred at reflux for 10–12 h (TLC-control). The mixture was evaporated to a volume of 10–20 mL, and the precipitate was filtered, washed with ether (2 × 50 mL), and dried under reduced pressure. M = 3.21 g. Yield 79%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 2.28 (s, 3 H), 6.72 (s, 1 H), 7.92 (d, J = 8.6 Hz, 1 H), 8.01 (d, J = 8.6 Hz, 1 H), 8.75 (s, 1 H), 10.34 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 12.8, 113.1, 118.0, 121.0 (q, J = 32.2 Hz), 122.5, 124.2 (q, J = 271.5 Hz), 127.8, 131.3, 133.7 (q, J = 2.2 Hz), 145.8 (q, J = 3.7 Hz), 153.9, 167.1.

5-(5-Chloropyridin-2-yl)-3-methyl-1H-pyrrole-2-carboxylic Acid (S16).

To a solution of S14 (5.7 g, 22 mmol, 1 equiv) in a mixture of dioxane–H₂O (1:1, 60 mL), NaOH (1.06 g, 25 mmol, 1.1 equiv) was added in one portion, and the reaction mixture was stirred at reflux for 10–12 h (TLC-control). Then sodium-salt solution was acidified by addition of an equivalent amount of HCl (12 M, 2.08 mL, 1.1 equiv), and the precipitate was filtered. M = 5.1 g. Yield 95%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 2.29 (s, 3 H), 6.70 (s, 1 H), 7.89 (d, J = 8.3 Hz, 1 H), 7.98 (d, J = 8.4 Hz, 1 H), 8.53 (s, 1 H), 11.28 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 12.8, 112.2, 120.2, 123.1, 126.9, 128.5, 132.2, 136.7, 147.8, 148.2, 163.1.

Thiazole-5-carbaldehyde (S17).

A solution of DMSO (16.96 g, 15.4 mL, 217 mmol, 2.5 equiv) in CH₂Cl₂ (100 mL) was added dropwise to a solution of oxalyl chloride (13.23 g, 8.94 mL, 104 mmol, 1.2 equiv) in CH₂Cl₂ (100 mL) at −70 to −80 °C. The resulting solution was stirred for 10 min, and a solution of alcohol (10 g, 87 mmol, 1 equiv) in CH₂Cl₂ (100 mL) was added dropwise at the same temperature. After 15 min, Et₃N (35.15 g, 48.3 mL, 347 mmol, 4 equiv) was added dropwise, and 5 min later, the reaction mixture was allowed to warm to rt. The reaction mixture was quenched with water (300 mL), and the layers were separated. The organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo (bath temperature not exceeding 45–50 °C). The purification by flash chromatography (hexanes/EtOAc, 3:1) afforded aldehyde as a slightly brown oil. M = 7.05 g. Yield = 72%.

¹H NMR (CDCl₃, 400 MHz).

δ = 8.48 (s, 1 H), 9.07 (s, 1 H), 10.04 (d, J = 1.0 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 139.4, 151.6, 160.1, 182.3.

5-Vinylthiazole (S18).

To the suspension of Ph₃P⁺MeI⁻ (25.74 g, 64 mmol, 1.1 equiv) in THF (70 mL), tBuOK (7.16 g, 64 mmol, 1.1 equiv) was added in one portion. The mixture was refluxed for 1 h and cooled to room temperature. Aldehyde S17 (6.55 g, 58 mmol, 1 equiv) in THF (50 mL) was added dropwise under cooling with an aqueous bath. The solvent was evaporated (bath temperature not exceeding 35 °C), and the residue was triturated in ether and filtered. The filtrate was evaporated, and the crude product was purified by flash chromatography using hexane-EtOAc mixture (3:1) as eluent (R_f = 0.5in hexane-EtOAc 3:1). M = 4.79 g. Yield = 74%.

¹H NMR (CDCl₃, 400 MHz).

δ = 5.29 (d, J = 10.9 Hz, 1 H), 5.57 (d, J = 17.3 Hz, 1 H), 6.82 (dd, J = 17.3, 10.9 Hz, 1 H), 7.74 (s, 1 H), 8.63 (s, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 117.2, 126.5, 138.0, 141.6, 151.7.

1-(Thiazol-5-yl)ethane-1,2-diol (S19).

To a solution of alkene S18 (4.7 g, 42 mmol, 1 equiv) in acetone-water (4:1, 50 mL), 4-methylmorpholine-4-oxide monohydrate (NMO monohydrate) (6.29 g, 47 mmol, 1.1 equiv) and potassium osmate(VI) dehydrate (0.16 g, 1 mol %) were added in one portion. The mixture was refluxed for 10–12 h (TLC-control) and cooled to room temperature. The solvent was evaporated, and crude product was purified by flash chromatography using pure EtOAc as eluent (R_f = 0.2 in EtOAc). M = 2.83 g. Yield = 46%.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 3.44–3.50 (m, 1 H), 3.51–3.58 (m, 1 H), 4.85 (q, J = 5.6 Hz, 1 H), 5.00 (t, J = 5.8 Hz, 1 H), 5.80 (d, J = 4.7 Hz, 1 H), 7.77 (s, 1 H), 8.96 (s, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 66.9, 68.0, 139.5, 142.0, 153.1.

5-(2,2,3,3,8,8,9,9-Octamethyl-4,7-dioxa-3,8-disiladecan-5-yl)-thiazole (S20).

To a solution of alcohol, S19 (2.83 g, 19 mmol, 1 equiv), and DMF (50 mL), imidazole (5.31 g, 78 mmol, 4 equiv) was added in one portion, followed by portionwise addition of tert-butyldimethylsilyl chloride (TBSCl) (8.81 g, 58 mmol, 3 equiv). The reaction mixture was stirred overnight at 50–60 °C, cooled to room temperature, diluted with water (100 mL), and extracted with EtoAc (3 × 50 mL). The combined organic phases were washed with water (50 mL) and brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to give an oil, which was purified by flash chromatography using Hexane-EtOAc (10:1) as eluent (R_f = 0.3 in 10:1 hexane-EtOAc). M = 5.87 g. Yield = 80%.

¹H NMR (CDCl₃, 400 MHz).

δ = −0.02–0.03 (m, 9 H), 0.10 (s, 3 H), 0.87 (s, 9 H), 0.89 (s, 9 H), 3.58 (dd, J = 9.9, 6.3 Hz, 1 H), 3.75 (dd, J = 9.9, 5.9 Hz, 1 H), 5.01 (t, J = 6.1 Hz, 1 H), 7.76 (s, 1 H), 8.73 (s, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = −5.4, −3;5.4, −4.9, −4.7, 18.3, 18.5, 25.8 (3C), 26.0 (3C), 69.2, 70.4, 139.6, 141.7, 152.6.

General Procedure E: For 1,2-Addition.

The appropriate thiazole (1.3 equiv) was dissolved in THF (1M) and cooled to −78 °C. At this temperature, n-BuLi (2.5 M, 1.4 equiv) was added dropwise under a nitrogen atmosphere. The reaction mixture was stirred for 20 min at −78 °C, and the appropriate imine (1 equiv) was added dropwise as a solution in THF (1 M). The reaction mixture was slowly (~1 h) warmed to 0 °C and poured into water (5 mL of water per 1 g of thiazole). The biphasic mixture was extracted with CH₂Cl₂ (3 × 100 mL). The combined organic phases were dried over Na₂SO₄, filtered, and evaporated to give a brown oil, which was purified by column chromatography. Eluent: hexanes/EtOAc (10:1, 5:1, 1:1, 0:1).

General Procedure F: For Amine Deprotection.

A 1 M HCl–MeOH solution was prepared by the dropwise addition of AcCl (1.5 equiv) to MeOH. The resulting solution was cooled to ambient temperature and added to a flask containing an appropriately protected compound (1 equiv). After dissolution, the reaction mixture was stirred for 1 h, evaporated, dissolved in CH₂Cl₂, and washed with 10% aqueous K₂CO₃. The organic layer was dried over Na₂SO₄, filtered, and evaporated, and the residue was loaded on silica. Eluting with CH₂Cl₂/MeOH (50:1) provided pure amine as a yellow oil.

General Procedure G: For TBDPS Protection.

Diol (1 equiv) was dissolved in CH₂Cl₂ (10 mL per 1 g), and imidazole (1.2 equiv) was added in one portion, followed by a portionwise addition of tert-butyl(chloro)diphenylsilane (TBDPSCl) (1.1 equiv). The reaction mixture was stirred overnight, diluted with water (10 mL per 1 g), and extracted with CH₂Cl₂ (3 × 50 mL). The combined organic phases were washed with brine and dried over anhydrous Na₂SO₄, filtered, and evaporated to give an oil, which was purified by flash chromatography using a hexane-EtOAc mixture (1:1 and 0:1) as eluent.

Allyl Allyl(2-amino-2-(4-(2-((tert-butyldiphenylsilyl)oxy)-1-hydroxyethyl)thiazol-2-yl)ethyl)carbamate (S23-fS and S23-fR).

Compounds S23-fS and S23-fR were obtained following in succession the general procedures E, F, and G from S20 (compounds S21 and S22 were considered pure enough and used directly in the next steps without any further purifications).

S23-fS. M = 2.94 g. Yield (over three steps) = 30%.

S23-fR. M = 1.81 g. Yield (over three steps) = 27%.

¹H NMR (CDCl₃, 400 MHz).

δ = 1.08 (s, 9 H), 2.43 (br. s., 3 H), 3.51–4.00 (m, 6 H), 4.40–4.54 (m, 1 H), 4.60 (d, J = 4.2 Hz, 2 H), 5.03 (dd, J = 7.1, 4.1 Hz, 1 H), 5.07–5.17 (m, 2 H), 5.20 (dd, J = 10.5, 1.3 Hz, 1 H), 5.29 (dd, J = 17.2, 1.4 Hz, 1 H), 5.67–5.83 (m, 1 H), 5.85–5.98 (m, 1 H), 7.36–7.49 (m, 6 H), 7.55 (s, 1 H), 7.61–7.68 (m, 4 H).

Methyl 2-(thiazol-4-yl)acetate (S24).

The compound (S24) was prepared by following a published procedure.⁶⁷

¹H NMR (CDCl₃, 400 MHz).

δ = 3.72 (s, 3 H), 3.89 (s, 2 H), 7.24 (d, J = 1.8 Hz, 1 H), 8.76 (d, J = 1.9 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 36.6, 52.2, 116.1, 149.5, 152.8, 170.7.

2-(Thiazol-4-yl)ethanol (S25).

A solution of ester S24 (0.481 mol) in THF (480 mL) was added dropwise to a suspension of LiAlH₄ (0.500 mmol, 1.0 equiv) in THF (480 mL) at 0 °C. The reaction mixture was stirred for 60 min at 0 °C. It was then quenched by successive addition of EtOAc (100 mL), water (37 mL), 10% NaOH (37 mL) solution, and water (74 mL) (the temperature should not exceed 0 °C). The precipitate was filtered and washed several times with THF. The filtrate was evaporated to give S25, which was used without further purification.

¹H NMR (CDCl₃, 400 MHz).

δ = 3.03 (t, J = 5.9 Hz, 2 H), 3.43 (br. s., 1 H), 3.93 (t, J = 5.9 Hz, 2 H), 7.04–7.05 (m, 1 H), 8.74 (d, J = 2.0 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 33.9, 61.5, 114.1, 152.8, 155.2.

2-(Thiazol-4-yl)ethylmethanesulfonate (S26).

To a solution of corresponding alcohol S25 (2.14 mmol) and triethylamine (0.36 mL, 2.59 mmol) in anhydrous dichloromethane (6 mL), kept to 0 °C, under an inert nitrogen atmosphere, was added mesyl chloride (2.31 mmol). The reaction was maintained at 0 °C during the first hour, followed by warming to room temperature, under vigorous stirring and nitrogen atmosphere for 3 h. Then, the solution was extracted with dichloromethane (3 × 30 mL), and the combined organic extracts were washed with a 10% aqueous HCl solution, washed with brine, dried over Na₂SO₄, filtered, and evaporated. The obtained residue was separated by chromatography on silica gel eluting with dichloromethane, followed by chloroform, to yield the desired O-mesylated derivative. Yield: 78%.

¹H NMR (CDCl₃, 400 MHz).

δ = 2.89 (s, 3 H), 3.24 (t, J = 6.5 Hz, 2 H), 4.56 (t, J = 6.5 Hz, 2 H), 7.13 (d, J = 1.3 Hz, 1 H), 8.76 (d, J = 1.8 Hz, 1 H).

4-Vinylthiazole (S27).

To a mixture of S26 (7.41 mmol) and triethylamine (TEA) (5 mL) in dichloromethane (DCM) (50 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (5 mL) slowly at 0 °C. The mixture was stirred at rt overnight and then diluted with 50 mL of DCM, washed with 2 N HCl three times and with brine once. The organic layer was dried over Na₂SO₄, filtered, and concentrated to dryness. The residue was purified by preparatory (prep) TLC to give S27. Yield: 58%.

¹H NMR (CDCl₃, 400 MHz).

δ = 5.39 (dd, J = 10.9, 1.5 Hz, 1 H), 6.09 (dd, J = 17.3, 1.5 Hz, 1 H), 6.77 (dd, J = 17.3, 10.9 Hz, 1 H), 7.14 (d, J = 1.8 Hz, 1 H), 8.76 (d, J = 1.8 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 114.9, 116.9, 129.5, 152.9, 155.1.

1-(Thiazol-4-yl)ethane-1,2-diol (S28).

To a solution of alkene S27 (42 mmol, 1 equiv) in acetone–water (4:1, 50 mL), NMO monohydrate (47 mmol, 1.1 equiv) and potassium osmate(VI) dehydrate (0.16 g, 1 mol %) were added in one portion. The mixture was refluxed for 10–12 h (TLC-control) and cooled to room temperature. The solvent was evaporated, and the crude product was purified by flash chromatography (eluent: EtOAc). Yield 70%.

¹H NMR (CDCl₃, 400 MHz).

δ = 3.77 (dd, J = 11.4, 7.2 Hz, 1 H), 3.91 (dd, J = 11.5, 3.4 Hz, 1 H), 4.72 (br. s., 1 H), 4.99 (dd, J = 6.9, 3.3 Hz, 1 H), 5.25 (br. s., 1 H), 7.32 (d, J = 1.9 Hz, 1 H), 8.71 (d, J = 2.0 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = 66.4, 71.3, 115.2, 153.6, 157.5.

4-(2,2,3,3,8,8,9,9-octamethyl-4,7-dioxa-3,8-disiladecan-5-yl)-thiazole (S29).

To a solution of alcohol, S28 (19 mmol, 1 equiv), and DMF (50 mL), imidazole (5.31 g, 78 mmol, 4 equiv) was added in one portion, followed by portionwise addition of TBSCl (8.81 g, 58 mmol, 3 equiv). The reaction mixture was stirred overnight at 50–60 °C, cooled to room temperature, diluted with water (100 mL), and extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with water (50 mL), washed with brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and evaporated to give an oil, which was purified by flash chromatography using hexane-EtOAc (10:1) as eluent. Yield = 78%.

¹H NMR (CDCl₃, 400 MHz).

δ = −0.01–0.04 (m, 9 H), 0.11 (s, 3 H), 0.86 (s, 9 H), 0.90 (s, 9 H), 3.68 (dd, J = 10.2, 7.2 Hz, 1 H), 3.93 (dd, J = 10.2, 3.8 Hz, 1 H), 5.03 (dd, J = 7.0, 3.6 Hz, 1 H), 7.28 (d, J = 1.9 Hz, 1 H), 8.75 (d, J = 2.1 Hz, 1 H).

¹³C NMR (CDCl₃, 100 MHz).

δ = −5.3, −5.2, −4.8, −4.5, 18.4, 18.5, 26.0 (3C), 26.1 (3C), 68.5, 73.8, 114.8, 152.3, 159.3.

Allyl N-Allyl-N-[2-[4-[1,2-bis[[tert-butyl(dimethyl)silyl]oxy]ethyl]-thiazol-2-yl]-2-(tert-butylsulfinylamino)ethyl]carbamate (S30).

Compounds S30-fR and S30-fS were obtained following the general procedure E.

Allyl N-Allyl-N-[2-amino-2-[4-(1,2-dihydroxyethyl)thiazol-2-yl]-ethyl]carbamate (S31).

Compounds S31-fR and S31-fS were obtained following the general procedure F.

Allyl N-Allyl-N-[2-amino-2-[4-[2-[tert-butyl(diphenyl)silyl]oxy-1-hydroxy-ethyl]thiazol-2-yl]ethyl]carbamate (S32).

Compounds S32-fR and S32-fS were obtained following the general procedure G.

The synthesis of compound S33 was reported earlier.¹⁰

1-(4-(((tert-Butyldimethylsilyl)oxy)methyl)thiazol-2-yl)ethanone (S34).

To a solution of thiazole S33 (49.06 g, 214 mmol, 1 equiv) in THF (210 mL), BuLi (2.5 M in hexane, 85.55 mL, 1.2 equiv) was added dropwise at −78 °C under argon atmosphere. After the end of the addition, the mixture was stirred for 10 min, and then the solution of N-methoxy-N-methyl-acetamide (24.26 g, 235 mmol, 1.1 equiv) in THF (50 mL) was added dropwise. The resulting mixture was stirred overnight and poured into a saturated solution of NH₄Cl (400 mL). The organic layer was separated, and the aqueous solution was extracted with CH₂Cl₂ (2 × 100 mL). The combined organic layers were washed with brine (200 mL), dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by flash chromatography using hexane–EtOAc (10:1) as eluent. M = 44.39 g. Yield = 76%.

¹H NMR.

(CDCl₃, 400 MHz) δ = 0.12 (s, 6 H), 0.94 (s, 9 H), 2.67 (s, 3 H), 4.90 (d, J = 1.1 Hz, 2 H), 7.54 (t, J = 1.1 Hz, 1 H).

¹³C NMR.

(CDCl₃, 100 MHz) δ = −5.3 (2C), 18.5, 26.0 (3C), 26.1, 62.2, 121.5, 159.7, 166.8, 191.8.

2-Amino-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)thiazol-2-yl)-propanenitrile (S35).

To the solution of S34 (44.39 g, 164 mmol, 1 equiv) in MeOH–NH₃ (330 mL), NaCN (16.0 g, 327 mmol, 2 equiv) and NH₄Cl (35.0 g, 654 mmol, 4 equiv) were added. The mixture was stirred for 4–5 d, and then water (600 mL) was added and extracted with DCM (3 × 200 mL). The combined organic layers were washed with brine (200 mL) and dried over Na₂SO₄, filtered, and concentrated. The obtained crude product was purified by flash chromatography using a hexane–EtOAc mixture (3:1) as eluent. The compound was obtained in racemic form. M = 16.46 g. Yield = 34%.

¹H NMR.

(CDCl₃, 400 MHz) δ = 0.12 (s, 6 H), 0.95 (s, 9 H), 1.92 (s, 3 H), 2.42 (br. s., 2 H), 4.86 (d, J = 1.2 Hz, 2 H), 7.21 (t, J = 1.2 Hz, 1 H).

¹³C NMR.

(CDCl₃, 100 MHz) δ = −5.3 (2C), 18.4, 26.0 (3C), 30.3, 52.6, 62.3, 114.8, 121.9, 158.2, 170.6.

2-(4-(((tert-Butyldimethylsilyl)oxy)methyl)thiazol-2-yl)propane-1,2-diamine (S36).

A solution of S35 (16.46 g, 55 mmol, 1 equiv) in Et₂O (55 mL) was added dropwise to a suspension of LiAlH₄ (6.31 g, 166 mmol, 3 equiv) in Et₂O (55 mL) at −10 °C. The reaction mixture was stirred for 12 h at 0 °C. It was then quenched by successive addition of water (7 mL), 10% NaOH (7 mL) solution, and water (7 mL) (the temperature should not exceed 0 °C). The precipitate was filtered and washed several times with Et₂O. The filtrate was evaporated to give diamine S36, which was purified by flash chromatography using pure EtOAc and CHCl₃–MeOH saturated with NH₃ (10:1) as eluents). M = 9.13 g. Yield = 55%.

¹H NMR.

(CDCl₃, 400 MHz) δ = 0.09 (s, 6 H), 0.92 (s, 9 H), 1.46 (s, 3 H), 1.61 (br. s., 4 H), 2.76 (d, J = 12.8 Hz, 1 H), 3.15 (d, J = 12.8 Hz, 1 H), 4.81 (d, J = 1.2 Hz, 2 H), 7.06 (t, J = 1.2 Hz, 1 H).

¹³C NMR.

(CDCl₃, 100 MHz) δ = −5.2 (2C), 18.5, 26.0 (3C), 28.1, 53.9, 58.1, 62.5, 113.5, 157.3, 179.8.

tert-Butyl (2-amino-2-(4-(((tert-butyldimethylsilyl)oxy)methyl)-thiazol-2-yl)propyl)carbamate (S37).

To a solution of diamine S36 (9.13 g, 30 mmol, 1 equiv) in CH₂Cl₂ (300 mL), a solution of Boc₂O (6.93 g, 32 mmol, 1.05 equiv) in CH₂Cl₂ (300 mL) was added dropwise at 0 °C. The mixture was stirred overnight, and then the solvent was evaporated. The crude product was used in the next step without purification. M = 12.1 g. Yield = 100%.

¹H NMR.

(CDCl₃, 400 MHz) δ = 0.11 (s, 6 H), 0.94 (s, 9 H), 1.42 (s, 9 H), 1.48 (s, 3 H), 1.85 (br. s., 2 H), 3.43–3.60 (m, 2 H), 4.80 (d, J = 1.2 Hz, 2 H), 5.07 (br. s., 1 H), 7.09 (t, J = 1.2 Hz, 1 H).

¹³C NMR.

(CDCl₃, 100 MHz) δ = −5.2 (2C), 18.5, 26.0 (3C), 28.2, 28.5 (3C), 51.6, 57.5, 62.4, 79.6, 114.2, 156.6, 157.1, 179.1.

General Procedure H: For Amide Coupling.

N,N-Diisopropylethylamine (DIPEA) (1 equiv) was added to an appropriate acid (1 equiv) followed by DMF (10 mL per 1 g of acid) and then N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-uronium hexafluorophosphate (HBTU) (1 equiv). The resulting solution was stirred for 10 min and added to a solution of appropriate amine (1 equiv) in DMF (10 mL per 1 g of amine) in several portions. The reaction mixture was stirred overnight; DMF was evaporated, and the residue was dissolved in DCM (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 Na₂SO₄, filtered, evaporated, and dry-loaded on silica. 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 I: for Deprotection.

To a solution containing protected compound (1 equiv) and N,N-dimethyl barbituric acid (NDMBA, 3 equiv) in MeOH (0.1 M solution), PPh₃ (10 mol %) was added under a nitrogen atmosphere followed by Pd(dba)₂ (5 mol %). The mixture was stirred for 1 d under reflux. After the mixture cooled, 50 mL of DCM 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 DCM/EtOH (~4:1, (2–4) × 50 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated. Purification by flash chromatography (eluent: DCM/MeOH (saturated with NH₃ ≈ 7 M), 10:1) afforded amine as a slightly brown or yellowish solid.

N-(2-Amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(5-chloropyridin-2-yl)-1H-pyrrole-2-carboxamide (1 and 2).

Compounds 1 and 2 were obtained following the general procedures H and I from amine S39 and acid S4b. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃–MeOH saturated with NH₃ (10:1 and 5:1).

1.M = 507 mg. Yield = 33% (over two steps). rt = 1.035 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 378 Da.

2.M = 335 mg. Yield = 22% (over two steps). rt = 1.022 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 378 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.68 (br. s., 2 H), 2.97 (dd, J = 13.2, 7.9 Hz, 1 H), 3.11 (dd, J = 13.3, 5.3 Hz, 1 H), 4.59 (d, J = 4.2 Hz, 2 H), 5.12–5.20 (m, 1 H), 5.46 (t, J = 5.3 Hz, 1 H), 6.88–6.94 (m, 2 H), 7.54 (s, 1 H), 7.89–8.01 (m, 2 H), 8.52–8.64 (m, 1 H), 8.80 (d, J = 7.5 Hz, 1 H), 11.89 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.8, 54.7, 55.8, 109.7, 114.3, 120.4, 128.4, 128.5, 133.4, 136.9, 139.1, 140.1, 147.6, 148.4, 160.0, 171.8.

High-resolution mass spectrometry (HRMS) electrospray ionization (ESI) calcd for C₁₆H₁₇ClN₅O₂S [M + H]⁺ 378.0786, found 378.0787.

N-(2-Amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(5-chloropyridin-2-yl)-3-methyl-1H-pyrrole-2-carboxamide (3 and 4).

Compounds 3 and 4 were obtained following the general procedures H and I from amine S39 and acid S16. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃–MeOH saturated with NH₃ (10:1 and 5:1).

3.M = 412 mg. Yield = 26% (over two steps). rt = 1.112 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 392 Da.

4.M = 284 mg. Yield = 18% (over two steps). rt = 1.140 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 392 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 2.31 (s, 3 H), 3.00 (dd, J = 13.2, 8.1 Hz, 1 H), 3.13 (dd, J = 13.3, 5.1 Hz, 1 H), 4.60 (s, 2 H), 5.13–5.23 (m, 1 H), 5.49 (br. s., 1 H), 6.76 (s, 1 H), 7.55 (s, 1 H), 7.82–7.87 (m, 1 H), 7.92–7.96 (m, 1 H), 8.59 (d, J = 2.3 Hz, 1 H), 8.76 (d, J = 7.5 Hz, 1 H), 11.80 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 13.1, 45.7, 54.2, 55.8, 111.9, 120.4, 123.5, 127.2, 128.4, 131.2, 136.9, 139.1, 140.1, 147.4, 148.3, 160.8, 172.0.

HRMS (ESI) calcd for C₁₇H₁₉ClN₅O₂S [M + H]⁺ 392.0942, found 392.0942.

N-(2-Amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (5 and 6).

Compounds 5 and 6 were obtained following the general procedures H and I from amine S39 and acid S4d. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃–MeOH saturated with NH₃ (10:1 and 5:1).

5.M = 262 mg. Yield = 38% (over two steps). rt = 1.260 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 412 Da.

6.M = 162 mg. Yield = 24% (over two steps). rt = 1.265 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 412 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 3.02 (dd, J = 13.2, 7.8 Hz, 1 H), 3.15 (dd, J = 13.4, 5.4 Hz, 1 H), 4.62 (s, 2 H), 5.15–5.26 (m, 1 H), 5.51 (br. s., 1 H), 6.99 (d, J = 3.9 Hz, 1 H), 7.05 (d, J = 3.9 Hz, 1 H), 7.56 (s, 1 H), 8.12 (d, J = 8.4 Hz, 1 H), 8.19 (dd, J = 8.6, 2.1 Hz, 1 H), 8.81–8.97 (m, 2 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.8, 54.7, 55.8, 111.2, 114.3, 118.9, 122.4 (q, J = 32.1 Hz), 124.0 (q, J = 271.5 Hz), 129.4, 133.1, 134.4 (q, J = 3.2 Hz), 139.1, 140.1, 146.0 (q, J = 4.0 Hz), 153.2, 159.9, 171.7.

HRMS (ESI) calcd for C₁₇H₁₇F₃N₅O₂S [M + H]⁺ 412.1050, found 412.1048.

N-(2-Amino-1-(5-(hydroxymethyl)thiazol-2-yl)ethyl)-3-methyl-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (7 and 8).

Compounds 8 and 7 were obtained following the general procedures H and I from amine S39 and the sodium salt S15. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃–MeOH saturated with NH₃ (10:1 and 5:1).

8.M = 358 mg. Yield = 40% (over two steps). rt = 1.202 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 426 Da.

7.M = 253 mg. Yield = 28% (over two steps). rt = 1.208 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 426 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.93 (br. s., 2 H), 2.33 (s, 3 H), 3.00 (dd, J = 13.2, 8.0 Hz, 1 H), 3.12 (dd, J = 13.3, 5.1 Hz, 1 H), 4.61 (s, 2 H), 5.13–5.22 (m, 1 H), 5.50 (br. s., 1 H), 6.91 (s, 1 H), 7.56 (s, 1 H), 8.00 (d, J = 8.4 Hz, 1 H), 8.19 (dd, J = 8.5, 2.1 Hz, 1 H), 8.80 (d, J = 7.2 Hz, 1 H), 8.89 (s, 1 H), 12.00 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 13.1, 45.7, 54.2, 55.8, 113.4, 119.0, 122.3 (q, J = 32.4 Hz), 124.0 (q, J = 271.8 Hz), 124.6, 127.3, 130.9, 134.4 (q, J = 3.3 Hz), 139.1, 140.1, 145.8 (q, J = 4.1 Hz), 153.1 (q, J = 1.1 Hz), 160.8, 171.8.

HRMS (ESI) calcd for C₁₈H₁₉F₃N₅O₂S [M + H]⁺ 426.1206, found 426.1216.

N-(2-Amino-1-(5-(hydroxymethyl)thiazol-2-yl)-2-methylpropyl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (9 and 10).

Compounds 9 and 10 were obtained following the general procedures H and I from amine S45 and acid S4d. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃–MeOH saturated with NH₃ (20:1 and 10:1).

9.M = 434 mg. Yield = 25% (over two steps). rt = 1.204 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 440 Da.

10.M = 252 mg. Yield = 15% (over two steps). rt = 1.218 min. Purity = 96%. LC-MS: m/z [M + H]⁺ = 440 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.10 (s, 3 H), 1.14 (s, 3 H), 4.63 (s, 2 H), 5.29 (d, J = 4.9 Hz, 1 H), 5.51 (br. s., 1 H), 6.96 (d, J = 3.9 Hz, 1 H), 7.05 (d, J = 3.8 Hz, 1 H), 7.58 (s, 1 H), 8.11 (d, J = 8.4 Hz, 1 H), 8.20 (dd, J = 8.4, 2.2 Hz, 1 H), 8.64 (d, J = 7.2 Hz, 1 H), 8.88–8.92 (m, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 27.6, 28.3, 52.8, 55.8, 59.7, 111.2, 115.1, 119.1, 122.4 (q, J = 32.2 Hz), 124.0 (q, J = 272.1 Hz), 129.3, 133.2, 134.5 (q, J = 3.2 Hz), 138.7, 140.3, 145.9 (q, J = 4.1 Hz), 153.3, 159.4, 169.9.

HRMS (ESI) calcd for C₁₉H₂₁F₃N₅O₂S [M + H]⁺ 440.1363, found 440.1359.

N-(2-Amino-1-(5-(hydroxymethyl)thiazol-2-yl)-2-methylpropyl)-3-methyl-5-(5-(trifluoro-methyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (11 and 12).

Compounds 11 and 12 were obtained following the general procedures H and I from amine S45 and the sodium salt S15. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃–MeOH saturated with NH₃ (20:1 and 10:1).

11.M = 609 mg. Yield = 32% (over two steps). rt = 1.268 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 454 Da.

12.M = 674 mg. Yield = 33% (over two steps). rt = 1.306 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 454 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.09 (s, 3 H), 1.14 (s, 3 H), 2.32 (s, 3 H), 4.63 (d, J = 3.5 Hz, 2 H), 5.28 (s, 1 H), 5.45–5.56 (m, 1 H), 6.91 (s, 1 H), 7.58 (s, 1 H), 8.00 (d, J = 8.4 Hz, 1 H), 8.19 (dd, J = 8.6, 2.2 Hz, 1 H), 8.50 (br. s., 1 H), 8.86–8.92 (m, 1 H), 12.24 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 13.4, 27.6, 28.1, 52.9, 55.8, 59.2, 113.6, 119.2, 122.3 (q, J = 32.3 Hz), 124.0 (q, J = 271.8 Hz) 124.5, 127.5, 131.0, 134.4 (q, J = 3.1 Hz), 138.7, 140.3, 145.8 (q, J = 4.1 Hz), 153.2, 160.4, 170.1.

HRMS (ESI) calcd for C₂₀H₂₃F₃N₅O₂S [M + H]⁺ 454.1519, found 454.1519.

N-(2-Amino-1-(5-(1,2-dihydroxyethyl)thiazol-2-yl)ethyl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (13 and 14).

Compounds 13 and were 14 obtained from amine S23 and acid S4d, following in sequence the general procedures H, I, and the TBDPS cleavage as described below. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃–MeOH saturated with NH₃ (10:1 and 5:1).

TBDPS cleavage: To a solution of TBDPS-protected compound (1 equiv) in THF (0.1M), a solution of tetra-n-butylammonium fluoride (TBAF) trihydrate (1.1 equiv) in THF (0.1M) was added in one portion. The mixture was stirred for 1–2 h at room temperature (TLC-control) and concentrated. Purification was done by flash chromatography using CHCl₃-MeOH saturated with NH₃ mixture (5:1 and 3:1) as eluent.

Note that compounds 13 & 14 were obtained as a diastereisomeric mixture of two single compounds having the absolute configuration of the chiral carbon a as fR for 13 and fS for 14.

13. M = 221 mg. Yield = 27% (over three steps). rt = 1.107 min. Purity = 97%. LC-MS: m/z [M + H]⁺ = 442 Da.

14. M = 485 mg. Yield = 41% (over three steps). rt = 1.160 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 442 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 2.99 (dd, 1 H), 3.13 (dd, J = 13.2, 5.2 Hz, 1 H), 3.42 (ddd, J = 10.7, 5.7, 2.4 Hz, 1 H), 3.50 (dd, J = 10.8, 6.1 Hz, 1 H), 4.75 (t, J = 5.8 Hz, 1 H), 4.96 (br. s., 1 H), 5.14–5.24 (m, 1 H), 5.71 (br. s., 1 H), 6.98 (d, J = 3.9 Hz, 1 H), 7.05 (d, J = 3.9 Hz, 1 H), 7.57 (s, 1 H), 8.13 (d, J = 8.4 Hz, 1 H), 8.20 (dd, J = 8.6, 2.1 Hz, 1 H), 8.83–8.93 (m, 2 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = (45.8, 45.8), 54.6, 66.7, (68.1, 68.1), 111.3, 114.3, 119.0, 122.4 (q, J = 32.3 Hz), 124.0 (q, J = 271.8 Hz), 129.4, 133.2, 134.5 (q, J = 3.3 Hz), (138.6, 138.6), (141.4, 141.4), 146.0 (q, J = 4.1 Hz), (153.2, 153.2), 159.9, (171.0, 171.1).

HRMS (ESI) calcd for C₁₈H₁₉F₃N₅O₃S [M + H]⁺ 442.1155, found 442.1155.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(5-chloropyridin-2-yl)-1H-pyrrole-2-carboxamide (15 and 16).

Compounds 15 and 16 were obtained following the general procedures H and I from amine S38 and acid S4b. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

15.M = 518 mg. Yield = 34% (over two steps). rt = 1.074 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 378 Da.

16.M = 381 mg. Yield = 25% (over two steps). rt = 1.079 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 378 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.73 (br. s., 2 H), 2.99 (dd, J = 13.2, 7.8 Hz, 1 H), 3.12 (dd, J = 13.2, 5.3 Hz, 1 H), 4.54 (s, 2 H), 5.17–5.24 (m, 1 H), 5.30 (br. s., 1 H), 6.85–6.97 (m, 2 H), 7.29 (s, 1 H), 7.89–8.00 (m, 2 H), 8.59 (s, 1 H), 8.83 (d, J = 7.7 Hz, 1 H), 11.91 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.8, 54.3, 59.8, 109.7, 114.2, 114.3, 120.4, 128.4, 128.6, 133.5, 136.9, 147.6, 148.4, 157.7, 160.0, 172.0.

HRMS (ESI) calcd for C₁₆H₁₇ClN₅O₂S [M + H]⁺ 378.0786, found 378.0786.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(5-fluoropyridin-2-yl)-1H-pyrrole-2-carboxamide (17 and 18).

Compounds 17 and 18 were obtained following the general procedures H and I from amine S38 and acid S4c. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

17.M = 420 mg. Yield = 31% (over two steps). rt = 0.987 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 362 Da.

18.M = 325 mg. Yield = 24% (over two steps). rt = 1.001 min. Purity = 96%. LC-MS: m/z [M + H]⁺ = 362 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.78 (br. s., 2 H), 2.99 (dd, J = 13.2, 7.8 Hz, 1 H), 3.12 (dd, J = 13.2, 5.3 Hz, 1 H), 4.54 (d, J = 3.0 Hz, 2 H), 5.17–5.24 (m, 1 H), 5.30 (br. s., 1 H), 6.84 (d, J = 3.9 Hz, 1 H), 6.92 (d, J = 3.8 Hz, 1 H), 7.28 (s, 1 H), 7.77 (td, J = 8.8, 2.9 Hz, 1 H), 7.99 (dd, J = 8.9, 4.4 Hz, 1 H), 8.55 (d, J = 2.9 Hz, 1 H), 8.80 (d, J = 7.8 Hz, 1 H), 11.82 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.9, 54.4, 59.8, 109.0, 114.1, 114.2, 120.6 (d, J = 4.6 Hz), 124.3 (d, J = 19.0 Hz), 128.0, 133.7, 137.0 (d, J = 24.0 Hz), 146.6 (d, J = 3.7 Hz), 157.7, 157.9 (d, J = 252.5 Hz), 160.1, 172.1.

HRMS (ESI) calcd for C₁₆H₁₇FN₅O₂S [M + H]⁺ 362.1081, found 362.1080.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(5-methylpyridin-2-yl)-1H-pyrrole-2-carboxamide (19 and 20).

Compounds 19 and 20 were obtained following the general procedures H and I from amine S38 and acid S4a. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

19.M = 357 mg. Yield = 35% (over two steps). rt = 0.829 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 358 Da.

20.M = 254 mg. Yield = 25% (over two steps). rt = 0.773 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 358 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.71 (br. s., 2 H), 2.30 (s, 3 H), 2.99 (dd, J = 13.2, 7.8 Hz, 1 H), 3.12 (dd, J = 13.2, 5.3 Hz, 1 H), 4.54 (s, 2 H), 5.16–5.25 (m, 1 H), 5.32 (br. s., 1 H), 6.81 (d, J = 3.8 Hz, 1 H), 6.90 (d, J = 3.8 Hz, 1 H), 7.29 (s, 1 H), 7.63 (dd, J = 8.2, 2.0 Hz, 1 H), 7.79 (d, J = 8.1 Hz, 1 H), 8.41 (d, J = 1.7 Hz, 1 H), 8.82 (d, J = 7.8 Hz, 1 H), 11.78 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 17.8, 46.0, 54.5, 59.8, 108.3, 114.0, 114.3, 118.7, 127.4, 131.0, 134.6, 137.4, 147.2, 149.2, 157.6, 160.0, 172.2.

HRMS (ESI) calcd for C₁₇H₂₀N₅O₂S [M + H]⁺ 358.1332, found 358.1337.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (21 and 22).

Compounds 21 and 22 were obtained following the general procedures H and I from amine S38 and acid S4d. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

21.M = 410 mg. Yield = 41% (over two steps). rt = 1.269 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 412 Da.

22.M = 312 mg. Yield = 32% (over two steps). rt = 1.266 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 412 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 3.00 (dd, J = 13.2, 7.8 Hz, 1 H), 3.14 (dd, J = 13.2, 5.3 Hz, 1 H), 4.55 (s, 2 H), 5.18–5.26 (m, 1 H), 5.32 (br. s., 1 H), 6.98 (d, J = 3.8 Hz, 1 H), 7.05 (d, J = 3.9 Hz, 1 H), 7.29 (s, 1 H), 8.13 (d, J = 8.4 Hz, 1 H), 8.20 (dd, J = 8.6, 1.7 Hz, 1 H), 8.84–8.96 (m, 2 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.9, 54.4, 59.8, 111.2, 114.1, 114.3, 118.9, 122.3 (q, J = 32.2 Hz), 124.0 (q, J = 272.2 Hz), 129.4, 133.1, 134.4 (q, J = 2.9 Hz), 146.0 (q, J = 3.7 Hz), 153.2, 157.7, 159.8, 171.8.

HRMS (ESI) calcd for C₁₇H₁₇F₃N₅O₂S [M + H]⁺ 412.1050, found 412.1056.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)-2-methylpropyl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (23 and 24).

Compounds 23 and 24 were obtained following the general procedures H and I from amine S44 and acid S4d. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (20:1 and 10:1).

23.M = 321 mg. Yield = 26% (over two steps). rt = 1.248 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 440 Da.

24.M = 208 mg. Yield = 17% (over two steps). rt = 1.234 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 440 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.08 (s, 3 H), 1.13 (s, 3 H), 4.57 (s, 2 H), 5.10–5.50 (m, 2 H), 6.96 (d, J = 3.9 Hz, 1 H), 7.05 (d, J = 3.9 Hz, 1 H), 7.32 (s, 1 H), 8.09 (d, J = 8.4 Hz, 1 H), 8.18 (dd, J = 8.6, 2.1 Hz, 1 H), 8.65 (d, J = 7.2 Hz, 1 H), 8.87–8.91 (m, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 27.5, 28.3, 52.8, 59.4, 59.8, 111.2, 114.4, 115.1, 119.1, 122.4 (q, J = 32.2 Hz), 124.0 (q, J = 271.6 Hz), 129.3, 133.2, 134.5 (q, J = 3.2 Hz), 145.9 (q, J = 4.1 Hz), 153.3, 157.3, 159.4, 169.9.

HRMS (ESI) calcd for C₁₉H₂₁F₃N₅O₂S [M + H]⁺ 440.1363, found 440.1359.

N-(2-Amino-1-(4-(2-hydroxyethyl)thiazol-2-yl)ethyl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (25 and 26).

Compounds and 25 and 26 were obtained following the general procedures H and I from amine S41 and acid S4d. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

25.M = 490 mg. Yield = 43% (over two steps). rt = 1.198 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 426 Da.

26.M = 368 mg. Yield = 32% (over two steps). rt = 1.153 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 426 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 2.85 (t, J = 6.9 Hz, 2 H), 3.01 (dd, J = 13.2, 7.8 Hz, 1 H), 3.15 (dd, J = 13.2, 5.1 Hz, 1 H), 3.71 (t, J = 6.8 Hz, 2 H), 4.69 (br. s., 1 H), 5.19–5.27 (m, 1 H), 6.99 (d, J = 3.8 Hz, 1 H), 7.05 (d, J = 3.8 Hz, 1 H), 7.18 (s, 1 H), 8.12 (d, J = 8.4 Hz, 1 H), 8.19 (dd, J = 8.4, 1.7 Hz, 1 H), 8.89 (s, 2 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 34.9, 46.0, 54.6, 60.2, 111.2, 114.1, 114.3, 118.9, 122.3 (q, J = 32.4 Hz), 124.0 (q, J = 271.8 Hz), 129.4, 133.1, 134.4 (q, J = 3.3 Hz), 146.0 (q, J = 4.2 Hz), 153.2 (q, J = 1.1 Hz), 154.1, 159.9, 171.3.

HRMS (ESI) calcd for C₁₈H₁₉F₃N₅O₂S [M + H]⁺ 426.1206, found 426.1204.

N-(2-Amino-1-(4-(3-hydroxypropyl)thiazol-2-yl)ethyl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (27 and 28).

Compounds 27 and 28 were obtained following the general proceduresH and I from amine S42 and acid S4d. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

27.M = 483 mg. Yield = 34% (over two steps). rt = 1.226 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 440 Da.

28.M = 727 mg. Yield = 39% (over two steps). rt = 1.220 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 440 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.73–1.83 (m, 2 H), 2.71 (t, J = 7.7 Hz, 2 H), 2.99 (dd, J = 13.2, 7.9 Hz, 1 H), 3.13 (dd, J = 13.2, 5.1 Hz, 1 H), 3.44 (t, J = 6.3 Hz, 2 H), 4.50 (br. s., 1 H), 5.17–5.24 (m, 1 H), 6.98 (d, J = 3.9 Hz, 1 H), 7.06 (d, J = 3.9 Hz, 1 H), 7.14 (s, 1 H), 8.13 (d, J = 8.5 Hz, 1 H), 8.21 (dd, J = 8.5, 2.1 Hz, 1 H), 8.84–8.94 (m, 2 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 27.6, 32.1, 46.0, 54.6, 60.2, 111.2, 113.1, 114.3, 118.9, 122.3 (q, J = 32.4 Hz), 124.0 (q, J = 271.8 Hz), 129.4, 133.1, 134.4 (q, J = 3.1 Hz), 146.0 (q, J = 4.2 Hz), 153.2, 156.6, 159.9, 171.5.

HRMS (ESI) calcd for C₁₉H₂₁F₃N₅O₂S [M + H]⁺ 440.1363, found 440.1377.

N-(2-Amino-1-(4-(1,2-dihydroxyethyl)thiazol-2-yl)ethyl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (29 and 30).

Compounds 29 and 30 were obtained from amine S32 and acid S4d, following in sequence the general procedures H, I, and the TBDPS cleavage (below). Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

TBDPS cleavage: To a solution of TBDPS-protected compound (1 equiv) in THF (0.1M), a solution of TBAF trihydrate (1.1 equiv) in THF (0.1M) was added in one portion. The mixture was stirred for 1–2 h at room temperature (TLC-control) and concentrated. Purification was done by flash chromatography using CHCl₃-MeOH saturated with NH₃ mixture (5:1 and 3:1) as eluent.

Note that compounds 29 & 30 were obtained as diastereisomeric mixture of two single compounds having the absolute configuration of the chiral carbon a as fR for 29 and fS for 30.

29.M = 358 mg. Yield = 29% (over two steps). rt = 1.143 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 442 Da.

30.M = 238 mg. Yield = 19% (over two steps). rt = 1.124 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 442 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 3.00 (ddd, J = 13.2, 7.8, 2.1 Hz, 1 H), 3.10–3.18 (m, 1 H), 3.49 (ddd, J = 10.9, 7.1, 2.0 Hz, 1 H), 3.71 (dt, J = 10.9, 4.1 Hz, 1 H), 4.65 (dd, J = 6.8, 4.2 Hz, 1 H), 4.72 (br. s., 1 H), 5.16–5.26 (m, 1 H), 5.35 (br. s., 1 H), 6.98 (d, J = 3.9 Hz, 1 H), 7.05 (d, J = 3.9 Hz, 1 H), 7.29–7.32 (m, 1 H), 8.13 (d, J = 8.4 Hz, 1 H), 8.20 (dd, J = 8.6, 2.1 Hz, 1 H), 8.84–8.94 (m, 2 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.7, 54.3, (65.8, 65.9), (71.3, 71.4), 111.3, 114.3, (114.5, 114.5), 118.9, 121.9 (q, J = 32.4 Hz), 124.0 (q, J = 271.8 Hz), 129.4, 133.2, 134.5 (q, J = 3.3 Hz), 146.0 (q, J = 4.2 Hz), 153.2 (q, J = 1.1 Hz), (158.4, 158.5), (159.9, 159.9), (171.5, 171.5).

HRMS (ESI) calcd for C₁₈H₁₉F₃N₅O₃S [M + H]⁺ 442.1155, found 442.1172.

N-(1-Amino-2-(4-(hydroxymethyl)thiazol-2-yl)propan-2-yl)-3-methyl-5-(5-(trifluoromethyl)-pyridin-2-yl)-1H-pyrrole-2-carboxamide (31).

Compound 31 (racemate) was obtained following the general procedures H and I from amine S37 and the sodium salt S15. The compound was purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (20:1 and 10:1).

M = 190 mg. Yield = 12%. rt = 1.271 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 440 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.74 (s, 3 H), 2.25 (s, 3 H), 2.92 (d, J = 13.2 Hz, 1 H), 3.12 (d, J = 13.2 Hz, 1 H), 4.51 (d, J = 5.1 Hz, 2 H), 5.26 (t, J = 5.7 Hz, 1 H), 6.88 (s, 1 H), 7.21–7.23 (m, 1 H), 7.99 (d, J = 8.4 Hz, 1 H), 8.18 (dd, J = 8.6, 2.3 Hz, 1 H), 8.28 (s, 1 H), 8.86–8.89 (m, 1 H), 12.02 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 13.1, 23.5, 51.8, 59.9, 60.5, 113.5, 113.8, 119.0, 122.2 (q, J = 32.3 Hz), 122.6, 125.4, 126.5, 130.6, 134.4 (q, J = 3.3 Hz), 145.8 (q, J = 4.1 Hz), 153.2 (q, J = 1.3 Hz), 156.8, 160.5, 176.6.

HRMS (ESI) calcd for C₁₈H₁₉F₃N₅O₂S [M + H]⁺ 440.1363, found 440.1373.

N-(1-Amino-2-(4-(hydroxymethyl)thiazol-2-yl)propan-2-yl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (32.

Compound 32 (racemate) was obtained following the general procedures H and I from amine S37 and acid S4d. The compound was purified using column chromatography on silica gel. The eluent was CHCl₃/MeOH saturated with NH₃ (10:1 and 5:1).

M = 332 mg. Yield = 38%. rt = 1.205 min. Purity = 98%. LC-MS: m/z [M + H]⁺ = 426 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.76 (s, 3 H), 2.94 (d, J = 13.3 Hz, 1 H), 3.14 (d, J = 13.3 Hz, 1 H), 4.52 (s, 2 H), 5.29 (br. s., 1 H), 6.90 (d, J = 3.9 Hz, 1 H), 7.04 (d, J = 3.9 Hz, 1 H), 7.23 (s, 1 H), 8.10 (d, J = 8.4 Hz, 1 H), 8.19 (dd, J = 8.6, 2.2 Hz, 1 H), 8.40 (s, 1 H), 8.85–8.92 (m, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 23.5, 51.7, 59.9, 60.6, 111.2, 113.8, 114.3, 118.9, 122.3 (q, J = 32.3 Hz), 124.0 (q, J = 271.8 Hz), 130.0, 132.9, 134.4 (q, J = 3.1 Hz), 145.9 (q, J = 4.2 Hz), 153.3, 156.9, 159.6, 176.4.

HRMS (ESI) calcd for C₁₈H₁₉F₃N₅O₂S [M + H]⁺ 426.1206, found 426.1217.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-3-methyl-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (33 and 34).

Compounds 33 and 34 were obtained following the general procedures H and I from amine S38 and sodium salt S15. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

33.M = 248 mg. Yield = 33% (over two steps). rt = 1.333 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 426 Da.

34.M = 162 mg. Yield = 22% (over two steps). rt = 1.328 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 426 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 2.33 (s, 3 H), 3.00 (dd, J = 13.2, 7.7 Hz, 1 H), 3.12 (dd, J = 13.3, 5.1 Hz, 1 H), 4.55 (s, 2 H), 5.18–5.26 (m, 1 H), 5.34 (br. s., 1 H), 6.90 (s, 1 H), 7.29 (s, 1 H), 7.98 (d, J = 8.6 Hz, 1 H), 8.17 (dd, J = 8.6, 2.2 Hz, 1 H), 8.81 (d, J = 6.8 Hz, 1 H), 8.86–8.90 (m, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 13.1, 46.1, 54.4, 59.8, 113.4, 114.0, 119.0, 122.3 (q, J = 32.2 Hz), 124.0 (q, J = 272.1 Hz), 124.6, 127.3, 130.9, 134.4 (q, J = 3.2 Hz), 145.8 (q, J = 4.1 Hz), 153.1, 157.7, 160.7, 172.2.

HRMS (ESI) calcd for C₁₈H₁₉F₃N₅O₂S [M + H]⁺ 426.1206, found 426.1213.

N-(2-Amino-1-(4,5-bis(hydroxymethyl)thiazol-2-yl)ethyl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (35 and 36).

Compounds 35 and 36 were obtained following the general procedures H and I from amine S40 and acid S4d. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (15:1 and 3:1).

35; (fR). M = 468 mg. Yield = 38% (over two steps). rt = 1.221 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 442 Da.

36; (fS). M = 290 mg. Yield = 24% (over two steps). rt = 1.225 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 442 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 2.99 (dd, J = 13.1, 7.9 Hz, 1 H), 3.07–3.15 (dd, J = 13.2, 5.3 Hz, 1 H), 4.46 (s, 2 H), 4.65 (s, 2 H), 5.08 (br. s., 1 H), 5.12–5.19 (m, 1 H), 5.45 (br. s., 1 H), 6.97 (d, J = 3.9 Hz, 1 H), 7.05 (d, J = 3.9 Hz, 1 H), 8.13 (d, J = 8.4 Hz, 1 H), 8.20 (dd, J = 8.6, 2.0 Hz, 1 H), 8.84–8.91 (m, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.5, 54.1, 55.1, 57.4, 111.2, 114.3, 118.9, 122.3 (q, J = 32.1 Hz), 124.0 (q, J = 271.5 Hz), 129.4, 133.1, 134.4 (q, J = 3.2 Hz), 136.3, 146.0 (q, J = 4.0 Hz), 150.8, 153.2, 159.8, 169.0.

HRMS (ESI) calcd for C₁₈H₁₉F₃N₅O₃S [M + H]⁺ 442.1155, found 442.1151.

N-(2-Amino-1-(thiazol-2-yl)ethyl)-5-(5-(trifluoromethyl)pyridin-2-yl)-1H-pyrrole-2-carboxamide (37 and 38).

Compounds 37 and 38 were obtained following the general procedures H and I from amine S43 and acid S4d. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

37; (fR). M = 220 mg. Yield = 30% (over two steps). rt = 1.230 min. Purity = 95%. LC-MS: m/z [M + H]⁺ = 382 Da.

38; (fS). M = 342 mg. Yield = 45% (over two steps). rt = 1.236 min. Purity = 96%. LC-MS: m/z [M + H]⁺ = 382 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 3.03 (dd, J = 13.2, 7.8 Hz, 1 H), 3.16 (dd, J = 13.2, 5.3 Hz, 1 H), 5.22–5.31 (m, 1 H), 6.98 (d, J = 3.8 Hz, 1 H), 7.06 (d, J = 3.8 Hz, 1 H), 7.61 (d, J = 3.2 Hz, 1 H), 7.77 (d, J = 3.2 Hz, 1 H), 8.13 (d, J = 8.5 Hz, 1 H), 8.20 (dd, J = 8.5, 2.0 Hz, 1 H), 8.80–9.01 (m, 2 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.9, 54.5, 111.2, 114.2, 118.9, 119.6, 122.3 (q, J = 32.4 Hz), 124.0 (q, J = 271.8 Hz), 129.3, 133.1, 134.4 (q, J = 3.3 Hz), 142.4, 145.9 (q, J = 4.1 Hz), 153.2 (q, J = 1.3 Hz), 159.8, 172.1.

HRMS (ESI) calcd for C₁₆H₁₅F₃N₅OS [M + H]⁺ 382.0944, found 382.0952.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(6-chloropyridin-3-yl)-1H-pyrrole-2-carboxamide (39 and 40).

Compounds 39 and 40 were obtained following the general procedures H and I from amine S38 and acid S4e. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

39; (fR). M = 375 mg. Yield = 24% (over two steps). rt = 1.044 min. Purity = 96%. LC-MS: m/z [M + H]⁺ = 378 Da.

40; (fS). M = 387 mg. Yield = 28% (over two steps). rt = 0.999 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 378 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.72 (br. s., 2 H), 2.99 (dd, J = 13.2, 7.9 Hz, 1 H), 3.13 (dd, J = 13.2, 5.3 Hz, 1 H), 4.52 (s, 2 H), 5.16–5.23 (m, 1 H), 5.28 (br. s., 1 H), 6.76 (d, J = 3.9 Hz, 1 H), 7.04 (d, J = 3.9 Hz, 1 H), 7.27 (s, 1 H), 7.51 (d, J = 8.5 Hz, 1 H), 8.25 (dd, J = 8.5, 2.6 Hz, 1 H), 8.61 (d, J = 8.1 Hz, 1 H), 8.86 (d, J = 2.4 Hz, 1 H), 12.07 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.5, 54.2, 59.8, 108.7, 112.8, 114.1, 124.2, 127.3, 128.3, 130.6, 135.3, 146.0, 147.8, 157.6, 160.4, 172.2.

HRMS (ESI) calcd for C₁₆H₁₇ClN₅O₂S [M + H]⁺ 378.0786, found 378.0786.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(6-(trifluoromethyl)pyridin-3-yl)-1H-pyrrole-2-carboxamide (41 and 42).

Compounds 41 and 42 were obtained following the general procedures H and I from amine S38 and acid S4f. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

41; (fR). M = 240 mg. Yield = 33% (over two steps). rt = 1.246 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 412 Da.

42; (fS). M = 290 mg. Yield = 40% (over two steps). rt = 1.262 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 412 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 3.03 (dd, J = 13.2, 7.8 Hz, 1 H), 3.16 (dd, J = 13.2, 5.4 Hz, 1 H), 4.55 (s, 2 H), 5.20–5.28 (m, 1 H), 5.36 (br. s., 1 H), 6.93 (d, J = 3.9 Hz, 1 H), 7.11 (d, J = 3.9 Hz, 1 H), 7.30 (s, 1 H), 7.88 (d, J = 8.3 Hz, 1 H), 8.47 (dd, J = 8.3, 1.7 Hz, 1 H), 8.71 (d, J = 7.8 Hz, 1 H), 9.22 (d, J = 1.6 Hz, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.6, 54.4, 59.8, 109.9, 113.0, 114.2, 120.8 (q, J = 2.6 Hz), 121.9 (q, J = 273.5 Hz), 129.2, 130.4, 130.9, 132.9, 143.7 (q, J = 33.9 Hz), 146.3, 157.7, 160.4, 172.1.

HRMS (ESI) calcd for C₁₇H₁₇F₃N₅O₂S [M + H]⁺ 412.1050, found 412.1054.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(6-(trifluoromethyl)pyridazin-3-yl)-1H-pyrrole-2-carboxamide (43 and 44).

Compounds 43 and 44 were obtained following the general procedures H and I from amine S38 and acid S4g. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

43; (fR). M = 322 mg. Yield = 42% (over two steps). rt = 1.095 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 413 Da.

44; (fS). M = 193 mg. Yield = 25% (over two steps). rt = 1.065 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 413 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 3.01 (dd, J = 13.1, 7.8 Hz, 1 H), 3.14 (dd, J = 13.2, 5.3 Hz, 1 H), 4.55 (s, 2 H), 5.20–5.28 (m, 1 H), 5.32 (br. s., 1 H), 7.03 (d, J = 3.9 Hz, 1 H), 7.22 (d, J = 3.9 Hz, 1 H), 7.30 (s, 1 H), 8.24 (d, J = 9.0 Hz, 1 H), 8.48 (d, J = 9.0 Hz, 1 H), 8.97 (d, J = 7.1 Hz, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.7, 54.3, 59.8, 112.7, 114.2, 114.4, 121.8 (q, J = 273.7 Hz), 124.0, 125.2 (q, J = 1.7 Hz), 130.1, 130.6, 148.1 (q, J = 33.9 Hz), 154.5, 157.7, 159.7, 171.6.

HRMS (ESI) calcd for C₁₆H₁₆F₃N₆O₂S [M + H]⁺ 413.1002, found 413.1000.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(5-chloropyrimidin-2-yl)-1H-pyrrole-2-carboxamide (45 and 46).

Compounds 45 and 46 were obtained following the general procedures H and I from amine S38 and acid S4h. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (10:1 and 5:1).

45; (fR). M = 186 mg. Yield = 24% (over two steps). rt = 1.078 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 379 Da.

46; (fS). M = 124 mg. Yield = 16% (over two steps). rt = 1.099 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 379 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 2.99 (dd, J = 13.2, 7.8 Hz, 1 H), 3.12 (dd, J = 13.3, 5.3 Hz, 1 H), 4.55 (s, 2 H), 5.16–5.26 (m, 1 H), 5.31 (br. s., 1 H), 6.91 (d, J = 3.8 Hz, 1 H), 6.99 (d, J = 3.8 Hz, 1 H), 7.29 (s, 1 H), 8.90 (s, 2 H), 8.97 (d, J = 7.2 Hz, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 46.1, 54.5, 59.8, 112.5, 114.1, 114.9, 127.3, 129.7, 132.2, 155.9 (2C), 156.3, 157.7, 159.5, 171.8.

HRMS (ESI) calcd for C₁₅H₁₆ClN₆O₂S [M + H]⁺ 379.0738, found 379.0745.

N-(2-Amino-1-(4-(hydroxymethyl)thiazol-2-yl)ethyl)-5-(2-methoxypyridin-4-yl)-1H-pyrrole-2-carboxamide (47 and 48).

Compounds 47 and 48 were obtained following the general procedures H and I from amine S38 and acid S8. Compounds were purified using column chromatography on silica gel. The eluent was CHCl₃-MeOH saturated with NH₃ (20:1 and 10:1).

47; (fR). M = 485 mg. Yield = 32% (over two steps). rt = 0.838 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 374 Da.

48; (fS). M = 423 mg. Yield = 28% (over two steps). rt = 0.839 min. Purity = 100%. LC-MS: m/z [M + H]⁺ = 374 Da.

¹H NMR (DMSO-d₆, 400 MHz).

δ = 1.74 (br. s., 2 H), 3.00 (dd, J = 13.1, 7.9 Hz, 1 H), 3.14 (dd, J = 13.2, 5.3 Hz, 1 H), 3.85 (s, 3 H), 4.54 (s, 2 H), 5.18–5.25 (m, 1 H), 5.30 (br. s., 1 H), 6.87 (d, J = 3.9 Hz, 1 H), 7.03 (d, J = 3.9 Hz, 1 H), 7.29 (t, J = 1.0 Hz, 1 H), 7.30 (d, J = 0.9 Hz, 1 H), 7.41 (dd, J = 5.5, 1.4 Hz, 1 H), 8.10 (d, J = 5.4 Hz, 1 H), 8.66 (d, J = 7.9 Hz, 1 H), 12.07 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz).

δ = 45.7, 53.2, 54.5, 59.8, 104.5, 109.8, 112.9, 113.1, 114.1, 128.7, 132.1, 141.5, 147.2, 157.6, 160.4, 164.5, 172.2.

HRMS (ESI) calcd for C₁₇H₂₀N₅O₃S [M + H]⁺ 374.1281, found 374.12.

Scheme 2.

Synthesis of 5-(2-Methoxypyridin-4-yl)-1H-pyrrole-2-carboxylic Acid

ABBREVIATIONS USED

HIV-1

human immunodeficiency virus type 1

Env

envelope

AIDS

acquire immunodeficiency syndrome

VSV-G

vesicular stomatitis virus-G

ADMET

absorption, distribution, metabolism, and excretion

TBDPSCl

tert-butyl(chloro)diphenylsilane

DCM

dichloromethane

DIPEA

N,N-diisopropylethylamine

HBTU

N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)-uronium hexafluorophosphate

NDMBA

N,N-dimethyl barbituric acid

TBSCl

tert-butyldimethylsilyl chloride

Alloc

allyloxycarbonyl