Design, synthesis and evaluation of small molecule CD4-mimics as entry inhibitors possessing broad spectrum anti-HIV-1 activity

Abstract

Since our first discovery of a CD4-mimic, NBD-556, which targets the Phe43 cavity of HIV-1 gp120, we and other groups made considerable progress in designing new CD4-mimics with viral entry-antagonist property. In our continued effort to make further progress we have synthesized twenty five new analogs based on our earlier reported viral entry antagonist, NBD-11021. These compounds were tested first in HIV-1 Env-pseudovirus based single-cycle infection assay as well as in a multi-cycle infection assay. Four of these new compounds showed much improved antiviral potency as well as cytotoxicity. We selected two of the best compounds 45A (NBD-14009) and 46A (NBD-14010) to test against a panel of 51 Env-pseudotyped HIV-1 representing diverse subtypes of clinical isolates. These compounds showed noticeable breadth of antiviral potency with IC₅₀ of as low as 150 nM. These compounds also inhibited cell-to-cell fusion and cell-to-cell HIV-1 transmission. The study is expected to pave the way of designing more potent and selective HIV-1 entry inhibitors targeted to the Phe43 cavity of HIV-1 gp120.

1. Introduction

Introduction of highly active anti-retroviral therapy (HAART), which uses a combination of drugs, made a remarkable impact in the AIDS epidemic by converting it to a manageable illness from once considered as a fatal disease. Despite this success more than 36 million people are living with AIDS and nearly 2 million people get newly infected each year (UNAIDS Global AIDS Update 2016). The major stumbling block in making any appreciable dent in such high HIV infection rate is drug resistance, side effects of the drugs and patient compliance. Moreover, none of the currently marketed drugs are curative of HIV. Therefore, there is an urgent need to develop new drugs, especially against targets critically important for HIV-1 life cycle. HIV-1 envelope glycoprotein is such a critical target. Soluble CD4 (sCD4) was first used to target gp120 and shown to inhibit a diverse strain of HIV and SIV¹. The clinical trial with sCD4 indicated that even at high dose level it failed to reduce the virus titer in a significant level². The only fusion inhibitor, Fuzeon (Enfuvirtide), a 36-residue based peptide drug that targets envelope glycoprotein gp41, was approved in 2003. Another entry inhibitor, Maraviroc, that targets the host coreceptor CCR5, was approved in 2007. Since then no entry/fusion inhibitors got approval for human use.

We reported the discovery of two small molecule HIV-1 inhibitors in 2005 by targeted screening of commercial libraries to the Phe43 cavity of HIV-1 envelope glycoprotein gp120³. Since then other groups also made considerable progress in designing inhibitors targeting the Phe43 cavity based on our earlier discovery^4-15. In 2012, we reported the co-crystal structure of NBD-556 bound to gp120 and confirmed that indeed this small molecule of 337 Da molecular weight binds to the Phe43 cavity¹⁶. However, later it was discovered that despite their anti-HIV-1 activity, this molecule also mimicked CD4⁸ and induced conformational changes in gp120 which may make it conducive to CCR5 coreceptor binding. In other words, this molecule behaves as viral entry agonist, an undesirable property of gp120 targeted drugs. We exploited the x-ray structural information of NBD-556 and other next generation NBD compounds bound to HIV-1 gp120¹⁷ to design next generation CD4-mimics with the goal of achieving viral entry antagonist property. We recently reported the structure-based design of NBD-11021 which showed not only much potent broadly neutralizing activity but also this molecule was experimentally confirmed to be a viral entry antagonist, a much desired property of this class of molecules¹⁸.

The x-ray structure of NBD-11021 bound to HIV-1 gp120 confirmed that this molecule binds to the Phe43 cavity¹⁸ but in a slightly different orientation than we observed with NBD-556 and other partial antagonists such as NBD-09027 so that the piperidine “N” forms a H-bond with Asp368, a critical interaction that was missing in all our compounds so far. The wide breadth of anti-HIV-1 activity profile of NBD-11021 against a large panel of HIV-1 pseudoviruses representing diverse subtypes of clinical isolates and availability of its structural information in complex with gp120 motivated us to continue the design of more robust and active analogs of this molecule.

Here we report the design, synthesis, biological evaluation and structure-activity relationships (SAR) of several new analogs of NBD-11021, some of which showed substantial improvement in antiviral activity and cytotoxicity profile. The study is expected to pave the way in designing more potent entry inhibitors with improved selectivity index for further preclinical studies.

2. Results and Discussion

2.1. Chemistry

The basic synthesis routes to make this series of NBD-compounds have been previously reported¹⁸. First, we prepared imine 2 from racemic pipecolic acid and racemic tert-butanesulfinamide (Scheme 1). Then two protected thiazoles 4 and 9 were prepared from 5-(2-hydroxyethyl)-4-methylthiazole and thiazole 10 was prepared as reported¹⁸. 1,2-addition of thiazoles 10 and 4 to imine 2 yielded a separable mixture of isomeric products 11A/B and 12A/B and addition of thiazole 9 gave a single stereoisomer 13B. After chromatographic purification the stereoisomers were separately used in the following steps. The synthetic intermediates derived from 11A or 12A have been labeled with an “A” letter in their numbering system (14A, 15A, etc.). The synthetic intermediates derived from 11B, 12B or 13B have been labeled with a “B” letter in the numbering system (14B, 15B, etc.). After acidic deprotection the amines were acylated with one of three commercially available 5-aryl-pyrrole-2-carboxylic acids (17-19). Finally Pd-catalyzed deprotection provided the target compounds 21-28. Compounds 21A and 22A and 21B and 22B were obtained from amine 14A and 14B, respectively, configurations of which were unambiguously determined in our previous work¹⁸. For the rest of the compounds stereochemistry was assigned based on analogy i.e., isomer with R,R or S,S configuration should be dominant product in the 1,2-addition to imine 2

Scheme-1.

Outline of the synthesis of intermediates and compounds 21A – 28B

For the preparation of imidazole-based NBD compounds, we relied on the same strategy (Scheme 2). However, the acidic N-H group of imidazole demanded an additional protecting group. After small experimentation we chose N,N-dimethylsulfamoyl group, because it can coordinate with Li, thus facilitating C-metalation of imidazole and it can be cleaved in one pot with TBS and SOtBu groups.

Scheme-2.

Outline of the synthesis of intermediates and compounds 29 – 37B

To this end imidazole 30 was prepared from (5-methyl-1H-imidazol-4-yl)methanol 29 as described^19;20 and treated with N,N-dimethylsulfamoyl chloride to yield a mixture of isomers 31A and 31B. The mixture of 31A and 31B was deprotonated with n-BuLi and imine 2 was added to give a mixture of eight enantiomeric pairs. This mixture was treated with MeOH-HCl to cleave three of four protecting groups. Fortunately, after deprotection stereoisomers can be separated, the isomer with higher R_f was marked as 33A and the isomer with lower R_f was marked as 33B. Unlike in the case of compound 14-16 no stereochemical assignment was made at this point. Amines 33A and 33B were separately converted into compounds 35-37A/B.

NBD-compounds without piperidine ring were prepared by a slightly modified route. First, amino acetaldehyde dimethyl acetal was protected with alloc and allyl groups and aldehyde functionality was unmasked by treatment with aqueous formic acid (Scheme 3). Then, enantiopure tert-butanesulfinamide was used to prepare both R- and S-imine 41. Separately, enantiomers of 41 were treated with metalated thiazole 10 to give a single addition product 42.

Scheme 3.

Outline of the synthesis of intermediates and compounds 45A (NBD-14009) – 47B

Compounds derived from (S)-41 and (R)-41 were marked as fS-42 and fR-42, respectively (Scheme 4). According to the Felkin-Ahn model the newly formed stereo center has opposite configuration than sulfur-stereo center. In other words compounds fS-43, fS-44, 45B, 46A, 47A have R-configuration, and compounds fR-43, fR-44, 45A, 46B, 47B have S-configuration. Since this was not confirmed unambiguously (by X-Ray analysis or other method) we prefer to use fS/fR markers for the sake of better reproducibility of this work.

Scheme-4.

Nomenclature fS was used to designate compounds obtained from (S)-isomers. Similarly fR was used for compounds synthesized from (R)-isomers.

As described in Scheme 3, three-step synthesis yielded compounds 45A - 47B. The enantiopurity of compounds 45A and 45B was measured using chiral column (77% and 88% respectively). We assume that stereochemical integrity is preserved during last three steps of the sequence and compounds 45A, 46A, 47A have the same enantiomeric excess (ee) value as the parent amine fS-43 (77%). The same implies for compounds 45B, 46B, 47B (ee=88%).

2.2. Biological Screening

2.2.1. Antiviral Screening of the new analogs by single-cycle and multi-cycle infectivity assays

We recently reported the successful conversion of a viral entry agonist NBD-556 to a viral entry antagonist NBD-11021¹⁸ by modifying regions II and III of the NBD-556 structure. Here we report the design of a new generation of analogs of NBD-11021 and evaluation of their HIV-1 inhibitory activity in a single-cycle and a multi-cycle infectivity assays (Table 1). We observed that compounds 21A to 28B did not show any improvement in antiviral activity or toxicity compared to NBD-11021A2 in both assays. It is noteworthy that introduction of a meta fluoro substituent in NBD-11021A2 resulted in very similar results in a single-cycle assay but yielded ~2-3-fold lower activity in the multi-cycle assay (IC₅₀ 0.85 μM for NBD-11021A2 compared to 1.6 and 2.6 μM for 21A and 21B, respectively). This is contrary to the report that introduction of a fluoro substituent in the same position of the phenyl ring of NBD-556 improved the antiviral activity ⁸. Earlier we have reported that introduction of a fluoro had mixed results²¹. However, it is noteworthy to mention that the introduction of a fluorine atom replacing hydrogen is known to improve potency or modulate physicochemical properties such as metabolic stability and pKa^22;23. The extension of the primary alcohol substituent (CH₂OH) to longer chains (CH₂CH₂OH and CH₂CH₂CH₂OH) resulted in substantial drop in antiviral activity. In order to understand the role of thiazole ring on the antiviral potency we replaced the thiazole ring with an imidazole ring but maintained other substituents. The imidazole ring containing compounds, 35A-37B, (Table 1) showed improvement in cytotoxicity levels but antiviral activity of all these compounds became worse in both assays. No specific structure-activity relationship (SAR) could be derived from this series. We also explored to alter piperidine ring to a simple amine because the x-ray structure of NBD-11021 bound to HIV-1 gp120 revealed that the hydrophobic part of the piperidine ring is located in the solvent exposed area, which is not desirable¹⁸. We used stereo selective synthesis to obtain the new series of NBD compounds. Compound 45A, where the Phenyl ring has only 4-Cl group, we observed that the antiviral activity is very similar to piperidine containing compound NBD-11021A2 [IC₅₀ 0f 2.2 μM vs 2.1 μM for 45A] in a single-cycle assay; however, cytotoxicity improved by about 2-fold, similar to the imidazole analogs. But in multi-cycle assay although the cytotoxicity improved, the antiviral activity decreased by about 3-fold. 45B, an isomer of 45A, showed poor activity. When we introduced a fluorine substitution in the ortho position at the phenyl ring of 45A the resultant compound 46A showed improvement in both antiviral and cytotoxic activities in both single-cycle and multi-cycle assays compared to its chloro analogs, 45A. 46B, the other isomer of 46A, showed very similar antiviral activity and cytotoxicity profiles to 46A in both single-cycle and multi-cycle antiviral assay. In this series we have observed that the introduction of fluorine in the phenyl ring helped in improving potency as discussed earlier. Replacing chloro in 46A by methyl yielded 47A which showed slightly less antiviral potency in a single-cycle assay. The isomer, 47B showed similar activity.

Table 1.

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

Compound ID	R1	R2	R3	TZM-bl Cells (Mean ± SD)		MT-2 Cells (Mean ± SD)
				IC50(μM)	CC50(μM )	IC50(μM)	cc50(μm )
NBD-11021A2	H	Cl	CH2OH	2.2 ± 0.2	23.6±0.2	0.85 ± 0.06	24.8±2.3
21A	F	Cl	CH2OH	3±0.4	15.2±0.2	1.6±0.2	17.2±0.4
21B	F	Cl	CH2OH	3.2±0.5	17.4±1.6	2.6±0.6	26±0.3
22A	F	CH3	CH2OH	2.7±0.2	18±0.6	3±0.6	16.1 ±0.4
22B	F	CH3	CH2OH	3.8±0.3	19.2±1.6	1.6±0.3	28.2±0.9
23A	F	CH3	CH3CH2OH	10.6±0.1	28.8±1	6.1 ±0.7	29.7±2
23B	F	CH3	CH3CH2OH	6.3±0.3	15.3±0.2	3±0.4	21.2±1.1
24A	F	Cl	CH3CH2OH	7.5±0.2	16.1 ±0.2	3.4±0.4	16.9±0.2
24B	F	Cl	CH3CH2OH	8±0.1	15.1 ±0.2	2.9±0.4	16.8±0.6
25A	H	Cl	CH3CH2OH	8.9±0.2	17.3±0.1	2±0.3	26.8±1.3
25B	H	Cl	CH3CH2OH	5.7±0.7	15.4±0.3	4.6±0.4	21 ±0.3
26B	F	CH3	CH3CH2CH2OH	7.9±1	17±0.7	7.4±0.4	20.6±1
27B	F	Cl	CH3CH2CH2OH	5.1 ±0.7	8.2±0.3	4.5±0.2	12.6±0.1
28B	H	Cl	CH3CH2CH2OH	7.6±0.8	13.7±1	5.8±1.3	19±0.2
35A	H	Cl	CH2OH	14.1 ±0.2	30±0.8	10.2±1.3	36.6±0.8
35B	H	Cl	CH2OH	15.8±0.6	33.8±0.8	18±3.6	45.2±1.4
36A	F	Cl	CH2OH	8.8±0.6	26.7±0.8	10.6±3.2	31.1±0.8
36B	F	Cl	CH2OH	16±0.3	30.7±1.3	22.8±2.8	36.4±2.5
37A	F	CH3	CH2OH	9.4±0.2	33.3±0.7	12.9±4.2	28.4±0.5
37B	F	CH3	CH2OH	16.9±0.5	34.5±0.7	22.3±4.7	37.1 ±2
45A (NBD-14009)	H	Cl	CH2OH	2.1 ±0.2	33.6±0.8	2.7±0.33	25.8±0.8
45B	H	Cl	CH2OH	6.5±1.6	34.1 ±3.9	2.1 ±0.04	28.2±0.5
46A (NBD-14010)	F	Cl	CH2OH	0.59±0.06	40.5±1.3	0.52±0.05	30.5±0.6
46B (NBD-14011)	F	Cl	CH2OH	0.68±0.03	26±2.5	0.77±0.01	22.7±0.6
47A (NBD-14012)	F	CH3	CH2OH	1.7±0.04	35±1.3	0.91±0.1	31.6±1.4
47B (NBD-14013)	F	CH3	CH2OH	1.1±0.1	26.2±1.3	0.65±0.06	24.3±1.5

^aThe reported IC₅₀ and CC₅₀ values represent the means ± standard deviations (SD) of three replicates

2.2.2. 45A and 46A are viral entry antagonists.

NBD-11021 represents our first example of a viral entry antagonist¹⁸. Since we made further chemical modifications to derive new analogs it was imperative to investigate whether the two best second generation compounds, 45A and 46A are also maintaining the viral entry antagonist trait. In order to verify we infected CD4-negative Cf2Th-CCR5+ cells with recombinant CD4-dependent HIV-1_ADA virus in the presence of varied concentrations of 45A and 46A. The viral entry agonist NBD-556 and the viral entry antagonist NBD-11021 were used as control (Figure 1). As expected, both compounds worked as viral entry antagonists. In other words, they did not enhance HIV-1 infectivity in the CD4 negative cells suggesting that the viral entry antagonist property was preserved in these compounds.

Figure 1. Infectivity of CD4-negative and CCR5-positive Cf2Th−CCR5 cells by CD4-dependent HIV-1_ADA.

Cf2Th−CCR5 cells were infected with CD4-dependent HIV-1_ADA in the presence of escalating concentrations of NBD-compounds. The relative virus infectivity indicates the amount of infection detected in the presence of the compounds divided by the amount of infection detected in the absence of the compounds. Three independent experiments were performed in triplicate, and the graph is representative of one experiment; the values represent the mean ± standard deviation.

2.2.3. Antiviral activity of 45A and 46A against a panel of HIV-1 Env pseudotyped reference viruses

We reported that NBD-11021 showed a broad range of antiviral activity against a large panel of Env-pseudotyped HIV-1 representing diverse subtypes of clinical isolates of different subtype. Here we present the results of these second generation compounds, 45A and 46A, against a panel of 51 Env-pseudotyped viruses including 13 recombinant HIV-1 clones, and we compared their antiviral activity with NBD-11021A2 (Table 2). Both, 45A and 46A exhibited improved anti-HIV-1 activity with respect to the first viral entry antagonist NBD-11021A2 (IC₅₀ in the range 0.32-4.4 μM, with the overall mean of 1.65 ± 0.13 μM). In fact, 45A was consistently active against all the pseudoviruses tested displaying an IC₅₀ in the range of 0.32-2.3 μM and the overall mean of 1.05 ± 0.07 μM. Furthermore, 46A showed a substantially improved anti-HIV-1 activity. The calculated IC₅₀s of this inhibitor were in the range of 0.15-0.87 μM and the overall mean was 0.46 ± 0.03 μM, with a nearly 4-fold improvement of the IC₅₀ with respect to NBD-11021A2. Finally, both 45A and 46A exhibited poor activity against the control pseudovirus VSV-G indicating that the inhibition of these compounds is more specific to HIV-1.

Table 2.

Antiviral activity of NBD-compounds against a panel of HIV-1 Env Pseudoviruses

			IC50 (μM) Mean ± S.D.a
Subtype	NIH #	Env	NBD-11021A2	45A (NBD- 14009)	46A (NBD- 14010)
A	11887	Q259env.w6	1.6±0.1c	1.3±0.02	0.74±0.06
	11888	QB726.70M.ENV.C4	1.3±0.3c	0.8±0.3	0.87±0.2
	11890	QF495.23M.ENV.A1	1.3±0.4c	1.2±0.3	0.62±0.04
	11891	QF495.23M.ENV.A3	0.88±0.4c	0.5±0.08	0.31±0.01
	11892	QF495.23M.ENV.B2	0.6±0.4c	0.74±0.1	0.52±0.07
		BG505-T332N	1±0.1c	0.78±0.2	0.33±0.04
		KNH1144	2.1±0.4c	0.67±0.02	0.43±0.2
A/D	11901	QA790.204I.ENV.A4	1.4±0.05c	2.1±0.3	0.62±0.02
	11903	QA790.204I.ENV.C8	1.5±0.02	0.74±0.06	0.21 ±0.003
	11904	QA790.204I.ENV.E2	0.71 ±0.09	0.47±0.09	0.4±0.05
A2/D	11905	QG393.60M.ENV.A1	0.41±0.2c	1.3±0.1	0.24±0.02
	11906	QG393.60M.ENV.B7	0.8±0.3c	0.41±0.04	0.35±0.01
	11907	QG393.60M.ENV.B8	0.48±0.2c	0.36±0.02	0.37±0.03
A/E	11603 (AE potential)	CRF01_AE clone 269	1.5±0.3c	2±0.2	0.7±0.02
		AA058	4.4±0.1	1±0.2	0.21 ±0.008
A/G	11601	CRF02_AG clone 263	1.4±0.3c	1.5±0.2	0.59±0.09
	11602	CRF02_AG clone 266	1±0.2c	1.6±0.2	0.68±0.02
	11605	CRF02_AG clone 278	1.3±0.5c	1.1±0.2	0.68±0.02
B		B41	0.68±0.1c	0.32±0.02	0.28±0.03
	11563	p1058_11.B11.1550b	1.1±0.8c	0.9±0.07	0.36±0.05
	11578	pWEAUd15.410.5017b	3±0.1c	0.67±0.1	0.28±0.04
	11018	QH0692, clone 42	0.52±0.1c	1.6±0.2	0.49±0.05
	11022	PVO, clone 4	1.9±0.1c	1.3±0.3	0.74±0.07
	11023	TRO, clone 11	1.2±0.05c	1.4±0.04	0.51 ±0.09
	11024	AC10.0, clone 29	0.32±0.01c	0.54±0.1	0.29±0.03
	11033	pWITO4160 clone 33	1.7±0.1c	1.1±0.2	0.5±0.06
	11035	pREJO4541 clone 67	1.6±0.3c	1±0.2	0.43±0.03
	11036	pRHPA4259 clone 7	1.4±0.2	0.86±0.3	0.32±0.01
	11037	pTHRO4156 clone 18	1±0.2c	0.82±0.08	0.69±0.08
	11038	pCAAN5342 clone A2	0.6±0.07c	0.39±0.02	0.22±0.02
	11058	SC422661.8	0.33±0.01c	0.54±0.02	0.2±0.01
C	11306	Du156, clone 12	2.2±0.5c	1.3±0.08	0.43±0.08
	11307	Du172, clone 17	1.9±0.5c	0.78±0.1	0.59±0.06
	11308	Du422, clone 1	3.3±0.4c	0.81±0.2	0.62±0.06
	11309	ZM197M.PB7	2±0.2	0.43±0.05	0.3±0.04
	11310	ZM214M.PL15	2.7±0.2c	1.1±0.1	0.42±0.03
	11311	ZM233M.PB6	1.7±0.1	1.1 ±0.1	0.21±0.02
	11313	ZM53M.PB12	1.5±0.2c	1.6±0.2	0.6±0.04
	11314	ZM109F.PB4	2.5±0.06	1.4±0.2	0.75±0.02
	11315	ZM135M.PL10a	2.7±1.2c	0.74±0.09	0.38±0.03
	11316	CAP45.2.00.G3	1.2±0.2	0.63±0.05	0.15±0.003
	11317	CAP210.2.00.E8	2.8±0.7c	0.76±0.05	0.43±0.05
	11502	HIV-16055-2, clone 3	2.3±0.1	1.2±0.2	0.86±0.08
	11908	QB099.391M.ENV.B1	1.7±0.4c	0.89±0.3	0.47±0.03
	11909	QB099.391M.ENV.C8	1.8±0.5c	1.3±0.2	0.49±0.05
D	11911	QA013.70I.ENV.H1	2.6±0.2c	2.2±1.6	0.66±0.04
	11912	QA013.70I.ENV.M12	2.2±0.3c	0.54±0.07	0.3±0.01
	11916	QD435.100M.ENV.B5	1.6±0.4	1.4±0.3	0.26±0.04
	11918	QD435.100M.ENV.E1	3.7±0.1c	0.52±0.3	0.46±0.09
D/A	11526 (mother)	MF535.W0M.ENV.C1	1.8±0.5c	1.5±0.3	0.33±0.02
	11517 (infant)	BF535.W6M.ENV.A1	2.8±0.6c	2.3±0.7	0.66±0.02
Control		VSV-G	7.1±0.7	6.3±0.6	8.7±1.1

^aThe reported IC₅₀ values represent the mean ± standard deviation (SD) of three replicates.

^bR5X4-tropic viruses; all the rest were CCR5-tropic viruses.

^cData previously published (Ref.18).

2.2.4. 45A and 46A prevent Cell-Cell fusion

CD4-gp120 interaction is critical in initiating the fusion of membranes of infected cells with neighboring cells inducing the formation of syncytia. Since these compounds target HIV-1 entry pathway, we investigated whether 45A and 46A could prevent HIV-1 Env-mediated cell-cell fusion. We cocultured MAGI-CCR5 cells with Env-expressing HL2/3 cells in the presence of different concentrations of NBD-compounds. NBD-11021A2 was used as control because we previously reported it to inhibit the cell-cell fusion process. As we expected, both, 45A and 46A inhibited the HIV-1 Env-mediated cell-to-cell fusion (Table 3).

Table 3.

Activity of NBD-compounds in cell-cell fusion assay and cell-to-cell HIV-1 transmission assay.

	NBD-11021A2 IC50 (μM) Mean ± S.D.	45A (NBD-14009) IC50 (μM) Mean ± S.D.	46A (NBD-14010) IC50 (μM) Mean ± S.D.
MAGI-CCR5/ HL2/3	8.9±0.4	11.9±2.4	15.2±2.4
GHOST (3) X4/R5 H9/HIV-1IIIb	8.8±0.6	8.6±0.3	6.4±0.9
GHOST (3) X4/R5 MOLT-4/CCR5/HIV- 1ADA	23±3	30±2	24±2.5

2.2.5. 45A and 46A inhibit cell-to-cell HIV-1 transmission

The cell-to-cell HIV-1 infection has been shown to be more efficient than the HIV-1 infection by cell-free virus, most likely because viruses are relatively protected from the environment and for the very high concentration of viral particles at the cellular contact sites ²⁴. We previously reported that NBD-11021 inhibits cell-to-cell HIV-1 transmission while NBD-556 failed to do so. Therefore, we tested the activity of 45A and 46A in cell-to-cell HIV-1 transmission assay. Ghost X4/R5 cells were used as target and H9/HIV-1_IIIB and MOLT-4/HIV-1_ADA as effector cells. Our results (Table 3) indicate that both 45A and 46A has similar activity to NBD-11021A2 against the cell-to-cell transmission of the CXCR4-tropic HIV-1 virus. Moreover, we detected similar but poor activity for 45A, 46A and NBD-11021A2 in the CCR5-tropic HIV-1 cell-to-cell transmission assay.

3. Conclusion

In this study we have reported the design, synthesis, antiviral activity and SAR of twenty five new analogs of NBD-11021A2, one of the best structure-based designed lead compounds that we reported earlier¹⁸. The antiviral assays in TZM-bl and MT-2 cells in Table 1 showed a good correlation (R² = 0.78; with p <0.0001). It was not possible to derive a correlation on the data in Table 2 because a diverse set of viruses were used. We also did not attempt to correlate these assays with the cell-cell fusion and virus transmission assays due to completely different nature of the assays. The SAR revealed that the thiazole ring in the series of compounds (21A – 28B and 45A – 47B), showed the best potent compounds when only CH₂OH substituent is present in the thiazole ring. However, when the thiazole ring was replaced by an imidazole ring, the activity dropped substantially although their cytotoxicity improved somewhat. The most dramatic improvement in antiviral activity and cytotoxicity were observed when we replaced the entire piperidine ring with a simple primary amine (CH₂NH₂). The best compound 46A also showed noticeable improvement in activity compared to NBD-11021A2 when tested against a large panel of pseudoviruses from a diverse set of Env from clinical isolates representing a wide variety of HIV-1 subtypes. 46A also inhibited cell-to-cell HIV-1 transmission and represents a substantially improved lead compound. It is important to mention that we are also aware that there is potential risk of development of resistance against these compounds since certain sections of gp120 are highly variable. Therefore, we are planning in depth studies on the resistance profile, if any, of these series of compounds. We anticipate that these studies and identification of substantially improved leads are expected to pave the way for designing more potent HIV-1 entry inhibitors.

4. Experimental

4.1. General

Commercial reagents and solvents were used without further purification. All reactions were performed in the air atmosphere unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) carried out on Merck TLC Silica gel plates (60 F254). We used UV light for visualization and basic aqueous potassium permanganate or iodine fumes as a developing agent. NMR (¹H and ¹³C) 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 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.

4.1.1. 5-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-methylthiazole (4)

4-Methyl-5-thiazoleethanol (10.0 g, 69.8 mmol) was dissolved in DMF (70 mL), imidazole (5.70 g, 83.8 mmol, 1.20 equiv) was added in one portion followed by portion wise addition of TBSCl (11.6 g, 77.0 mmol, 1.10 equiv). The reaction mixture was stirred overnight at 50-60 °C, cooled to r.t., diluted with water (0.5 L) and extracted with hexane (3×100 mL). The combined organic phases were dried over Na₂SO₄, and evaporated to give an oil which was purified by distillation at reduced pressure. bp:115 - 120 °C (1-2 torr). Yield:82% (14.71 g).

¹H NMR (CDCl₃, 400 MHz): δ = 0.03 (s, 6H), 0.90 (s, 9H), 2.41 (s, 3H), 2.97 (t, J = 6.4 Hz, 2H), 3.79 (t, J = 6.4 Hz, 2H), 8.57 (s, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = -5.3 (2C), 15.1, 18.4, 26.0 (3C), 30.0, 63.4, 128.2, 149.3, 149.7.

4.1.2. 5-(2-chloroethyl)-4-methylthiazole (5)

4-Methyl-5-thiazoleethanol (39.0 g, 272 mmol) was dissolved in CHCl₃ (270 mL) and SOCl₂ (40 mL, 0.55 mol, ~2 equiv) was added dropwise with cooling on a water bath. The mixture was refluxed for 4 hours, cooled to r.t. and evaporated. The residue was suspended in CH₂Cl₂ and aqueous K₂CO₃ (38 g, 0.28 mol, ~ 1 equiv in 200 ml H₂O). After stirring for 10 minutes the organic layer was separated and the aqueous layer was extracted with CH₂Cl₂ (2×100 mL). The combined organic layers were dried over Na₂SO₄ and evaporated. The residue was purified by distillation at reduced pressure. bp: 77 - 78 °C (1-2 torr.); Yield: 84% (36.97 g).

¹H NMR (CDCl₃, 400 MHz): δ = 2.37 (s, 3H), 3.17 (t, J = 7.0 Hz, 2H), 3.62 (t, J = 7.0 Hz, 2H), 8.56 (s, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = 14.9, 29.6, 44.2, 127.0, 149.9, 150.2.

4.1.3. 3-(4-methylthiazol-5-yl)propanenitrile (6)

NaCN (11.06, 0.226 mol) was suspended in DMF and 5-(2-chloroethyl)-4-methylthiazole (36.50 g, 0.226 mole) was added and the mixture was stirred for 8 hours at r.t. and then at 60 - 80 °C. The reaction mixture was cooled to r.t., diluted with (0.5 L) and extracted with CH₂Cl₂ (3×100 mL). The combined organic layers were dried over Na₂SO₄ and evaporated. The residue was pure enough for the next step. Yield: 98% (33.55 g).

¹H NMR (CDCl₃, 400 MHz): δ = 2.40 (s, 3H), 2.60 (t, J = 7.2 Hz, 2H), 3.11 (t, J = 7.2 Hz, 2H), 8.59 (s, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = 14.9, 19.4, 22.5, 118.3, 127.1, 150.2, 150.3.

4.1.4. 3-(4-Methyl-thiazol-5-yl)-propionic acid methyl ester (7)

3-(4-methylthiazol-5-yl)propanenitrile (33.52 g, 0.22 mol) was dissolved in MeOH (1 L), H₂SO₄ (90 mL, 1.69 mol, 7.7 equiv) was added dropwise and the reaction mixture was refluxed for two weeks (~8 hours a day). The reaction mixture was evaporated to 1/3 of a volume and poured into aqueous K₂CO₃ (276 g, 2 mol, in 0.5 L H₂O) solution and CH₂Cl₂ (500 mL). After stirring for 10 minutes the organic layer was separated and the aqueous layer was extracted with CH₂Cl₂ (2×100 mL). The combined organic layers were dried over Na₂SO₄ and evaporated. The residue was purified by means of liquid chromatography (eluent: hexanes/EtOAc, 10:1, 5:1). The upper spot is the ester the second spot is a starting material (M_RCN=3.50 g).

Yield: 81% ( 29.51 g; brsm).

¹H NMR (CDCl₃, 400 MHz): δ = 2.42 (s, 3H), 2.64 (t, J = 7.5 Hz, 2H), 3.11 (t, J = 7.5 Hz, 2H), 3.70 (s, 3H), 8.58 (s, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = 14.8, 21.6, 35.5, 51.8, 129.6, 149.2, 149.4, 172.3.

4.1.5. 3-(4-methylthiazol-5-yl)propan-1-ol (8)

A solution of 3-(4-methyl-thiazol-5-yl)-propionic acid methyl ester (29.51 g, 0.160 mol) in THF (160 mL) was added dropwise to a suspension of LiAlH₄ (6.10 g, 0.160 mmol) in THF (160 mL). The reaction mixture was stirred for 1 hour, and quenched by successive addition of water (6 mL), 10 % NaOH (6 mL) solution and water (12 mL). The precipitate was filtered and washed several times with THF. The filtrate was evaporated to give title compound, which was used without purification. Yield: 87% ( 22.02 g).

¹H NMR (CDCl₃, 400 MHz): δ = 1.79 - 1.92 (m, 2H), 2.36 (s, 3H), 2.85 (t, J = 7.6 Hz, 2H), 3.06 (br. s, 1H), 3.65 (t, J = 6.2 Hz, 2H), 8.52 (s, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = 14.7, 22.6, 34.3, 61.1, 131.6, 148.5, 149.1.

4.1.6. 5-(3-((tert-butyldimethylsilyl)oxy)propyl)-4-methylthiazole (9)

3-(4-methylthiazol-5-yl)propan-1-ol (22.02 g, 140 mmol) was dissolved in DMF (140 mL), imidazole (12.40 g, 182 mmol, 1.20 equiv) was added in one portion followed by portion wise addition of TBSCl (25.3 g, 168 mmol, 1.10 equiv). The reaction mixture was stirred overnight at 50-60 °C, cooled to r.t., diluted with water (0.5 L) and extracted with hexane (3×100 mL). The combined organic phases were dried over Na₂SO₄, and evaporated to give an oil which was purified by distillation at reduced pressure. bp:bp: 120 °C (2 torr). Yield: 77% (29.16 g).

¹H NMR (CDCl₃, 400 MHz): δ = 0.06 (s, 6H), 0.91 (s, 9H), 1.75 - 1.86 (m, 2H), 2.39 (s, 3H), 2.85 (t, J = 7.5 Hz, 2H), 3.64 (t, J = 6.0 Hz, 2H), 8.55 (s, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = -5.2 (2C), 14.9, 18.4, 22.5, 26.0 (3C), 34.6, 61.7, 131.6, 148.7, 148.9.

4.1.7. General procedure for 1,2-addition reaction.

Appropriate thiazole (4, 9 or 10; 1.3 equiv) was dissolved in THF (1 M) and cooled to −78 °C. At this temperature n-BuLi (2.5 M, 22 mL, 55 mmol, 1.4 equiv) was added dropwise under the nitrogen atmosphere. The reaction mixture was stirred for 30 minutes at −78 °C, and 2 (1 equiv) was added dropwise as a solution in THF (1 M). The reaction mixture was slowly (~1 hour) warmed to 0 °C, and poured into saturated NH₄Cl aqueous solution (volume equals to that of the reaction mixture). The biphasic mixture was extracted with CH₂Cl₂ (3×100 mL). The combined organic phases were dried over Na₂SO₄, and evaporated to give a brown oil which was purified by means of column chromatography.

The upper spot on TLC (EtOAc) is thiazole, then imine (if present) then two reaction products. Compounds 11A and 11B were synthesized as per the method reported earlier¹⁸.

4.1.8. allyl 2-((5-(2-((tert-butyldimethylsilyl)oxy)ethyl)-4-methylthiazol-2-yl)(1,1-dimethylethylsulfinamido)methyl)piperidine-1-carboxylate (12)

Eluent: hexanes/EtOAc, 3:1, then 1:1, then pure EtOAc.

First fraction: (mixture):10.19 g. Second fraction: 12A:2.13 g; Third fraction: 12B:11.04 g.

Second fraction (12A): Yield: 15% (2.13 g). R_f=0.53 (hexanes/EtOAc, 1:1)

¹H NMR: (CDCl₃, 400 MHz) δ = 0.03 (s, 6H), 0.89 (s, 9H), 1.19 (s, 6H), 1.24 (s, 3H), 1.40 - 1.88 (m, 9H), 2.34 (s, 3H), 2.93 (t, J = 6.1 Hz, 2H), 3.78 (t, J = 6.1 Hz, 2H), 4.42 - 4.53 (m, 1H), 4.58 - 4.80 (m, 2H), 5.08 (d, J = 9.8 Hz, 1H), 5.24 (d, J = 9.8 Hz, 1H), 5.35 (d, J =7.1 Hz, 1H), 5.90 - 6.06 (m, 1H).

Third fraction (12B): Yield: 42% (11.04 g). R_f=0.33 (hexanes/EtOAc, 1:1);

¹H NMR: (CDCl₃, 400 MHz) δ = 0.03 (s, 6H), 0.89 (s, 9H), 1.24 (s, 6H), 1.28 (s, 3H), 1.34 - 1.96 (m, 7H), 2.31 (s, 3H), 2.89 (t, J = 6.4 Hz, 2H), 3.75 (t, J = 6.4 Hz, 2H), 3.87 - 4.07 (m, 1H), 4.24 - 4.54 (m, 3H), 4.58 - 4.91 (m, 1H), 4.97 (dd, J = 7.8, 5.6 Hz, 1H), 5.16 (d, J = 10.5 Hz, 1H), 5.20 (d, J = 17.5 Hz, 1H), 5.63 - 6.05 (m, 1H).

4.1.9. allyl 2-((5-(3-((tert-butyldimethylsilyl)oxy)propyl)-4-methylthiazol-2-yl)(1,1-dimethylethylsulfinamido)methyl)piperidine-1-carboxylate (13B)

Only one diastereomer was formed in this reaction.

Eluent: hexanes/EtOAc, 3:1, then 1:1, then pure EtOAc; R_f=0.52 (EtOAc). Yield: 56% (5.35 g).

¹H NMR: (CDCl₃, 400 MHz) δ = 0.05 (s, 6H), 0.90 (s, 9H), 1.22 (s, 6H), 1.27 (s, 3H), 1.39 - 1.83 (m, 7H), 1.85 - 2.01 (m, 1H), 2.10 - 2.38 (m, 1H), 2.18 (s, 3H), 2.28 (s, 2H), 2.76 (t, J = 7.3 Hz 1H), 3.62 (t, J = 6.1 Hz, 2H), 3.85 - 4.08 (m, 1H), 4.16 - 4.57 (m, 3H), 4.95 (dd, J = 8.2, 5.5 Hz, 1H), 5.08 - 5.23 (m, 2H), 5.62 - 5.88 (m, 1H).

Instead a second isomer admixture 1-(5-(3-((tert-butyldimethylsilyl)oxy)propyl)-4-methylthiazol-2-yl)hexahydroimidazo[1,5-a]pyridin-3(2H)-one was isolated from the reaction mixture (R_f=0.40 (EtOAc), Yield = 32% (2.20 g).

¹H NMR: (CDCl₃, 400 MHz) δ = 0.06 (s, 6 H), 0.91 (s, 9 H), 1.19 - 1.34 (m, 2 H), 1.58 - 1.74 (m, 2 H), 1.74 - 1.85 (m, 2 H), 1.87 - 1.95 (m, 1 H), 1.98 - 2.04 (m, 1 H), 2.31 (s, 3 H), 2.69 (td, J=12.5, 3.2 Hz, 1 H), 2.79 (t, J=7.6 Hz, 2 H), 3.42 - 3.49 (m, 1 H), 3.64 (t, J=5.9 Hz, 2 H), 3.88 - 3.95 (m, 1 H), 4.56 (dd, J=6.5, 1.8 Hz, 1 H), 5.21 (br. s, 1 H).

4.1.10. General procedure for amine deprotection.

1M HCl-MeOH (5 - 20 equiv) solution was prepared by dropwise addition of AcCl to MeOH under a water bath cooling system. The resulting solution was cooled to an ambient temperature and added to a flask containing fully protected compound (11, 12 or 13; A or B isomers). After dissolution the reaction mixture was stirred for 1 hour, evaporated in vacuo (no heating). The residue was dissolved in CH₂Cl₂ and washed with 10% aqueous K₂CO₃ solution. The organic layer was separated and the aqueous layer was extracted with CH₂Cl₂ (2×100 mL). The combined organic layers were dried over Na₂SO₄ and evaporated. The residue was purified by means of chromatography. Eluent: CH₂Cl₂/MeOH (50:1, 10:1).

Compounds 14A and 14B were prepared as reported previously¹⁸.

4.1.11. allyl 2-(amino(5-(2-hydroxyethyl)-4-methylthiazol-2-yl)methyl)piperidine-1-carboxylate

First fraction (15A). Yield: 70% (909 mg).

¹H NMR: (CDCl₃, 400 MHz) δ = 1.39 - 1.77 (m, 7H), 2.18 (br. s, 3H), 2.34 (s, 3H), 2.98 (t, J = 6.4 Hz, 2H), 2.79 - 3.05 (br. s, 1H), 3.82 (t, J = 6.4 Hz, 1H), 4.00 - 4.33 (m, 1H), 4.33 - 4.51 (m, 1H), 4.52 - 4.58 (m, 1H), 4.65 (d, J = 4.2 Hz, 2H), 5.23 (dd, J = 10.5, 1.3 Hz, 1H), 5.35 (d, J = 17.6 Hz, 1H), 5.92 - 6.05 (m, 1H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 15.1, 19.4, 23.3, 24.6, 30.0, 30.2, 40.9, 59.0, 62.7, 62.8, 117.5, 129.0, 133.1, 149.1, 159.9, 168.4.

Second fraction (15B). Yield: 41% (1.65 g).

¹H NMR: (CDCl₃, 400 MHz) δ = 1.38 - 1.57 (m, 1H), 1.56 - 1.74 (m, 4H), 2.05 (br. s, 3H), 2.16 - 2.22 (m, 1H), 2.30 (s, 3H), 2.92 (t, J = 6.1 Hz, 2H), 3.06 (t, J = 13.1 Hz, 1H), 3.77 (t, J = 6.2 Hz, 2H), 3.99 - 4.10 (m, 1H), 4.21 - 4.34 (m, 2H), 4.34 - 4.42 (m, 1H), 4.51 (d, J = 10.0 Hz, 1H), 5.14 (dd, J = 10.5, 1.2 Hz, 1H), 5.19 (dd, J = 17.2, 1.5 Hz, 1H), 5.73 - 5.86 (m, 1H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 15.0, 19.1, 25.2, 25.4, 30.0, 40.5, 52.4, 57.1, 63.1, 66.0, 117.1, 127.8, 133.2, 148.1, 155.4, 163.9.

4.1.12. allyl 2-(amino(5-(3-hydroxypropyl)-4-methylthiazol-2-yl)methyl)piperidine-1-carboxylate (16B)

Yield: 68% (1.84 g). R_f=0.36 (CHCl₃/MeOH, 7:1).

¹H NMR: (CDCl₃, 400 MHz) δ = 1.36 - 1.53 (m, 1H), 1.54 - 1.72 (m, 4H), 1.76 - 1.85 (m, 2H), 1.94 - 2.22 (m, 4H), 2.26 (s, 3H), 2.77 (t, J = 7.5 Hz, 2H), 3.03 (t, J = 12.4 Hz, 1H), 3.62 (t, J = 6.3 Hz, 2H), 4.04 (d, J = 12.0 Hz, 1H), 4.23 - 4.33 (m, 2H), 4.39 (dd, J = 13.3, 5.4 Hz, 1H), 4.49 (d, J = 10.0 Hz, 1H), 5.11 (dq, J = 10.5, 1.4 Hz, 1H), 5.16 (dq, J = 17.2, 1.6 Hz, 1H), 5.69 - 5.84 (m, 1H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 14.8, 19.0, 22.7, 25.1, 25.4, 34.2, 40.3, 52.3 (br.), 56.7, 61.2, 65.9, 117.0, 131.3, 133.1, 146.8, 155.2, 169.5.

4.1.13. General procedure for amide coupling.

To a suspension of appropriate aryl-1H-pyrrole-2-carboxylic acid (17, 18 or 19; 1 equiv) in DMF (10 mL per 1 g of acid) DIPEA (1 equiv) was added followed by HBTU (1 equiv). The resulting solution was stirred for 10 minutes followed by the addition of the appropriate amine (1 equiv) in DMF (10 mL per 1 g of amine). The reaction mixture was stirred overnight and most of the DMF was evaporated. The residue was dissolved in CH₂Cl₂ and washed with 5 % aqueous NaOH solution. The organic layer was separated and the aqueous layers were extracted with CH₂Cl₂ (2x50 mL). The combined organic layer was dried over Na₂SO₄ and evaporated. The residue was purified by means of chromatography. Eluent: EtOAc or CH₂Cl₂/MeOH (50:1). The collected material was used in the next step without analysis.

4.1.14. General procedure for deprotection.

To a solution containing alloc-protected compound (1 mmol) and N,N'-dimethylbarbituric acid (3 mmol) in methanol (10 mL), PPh₃ (10 mol. %) was added under a nitrogen atmosphere followed by Pd(dba)₂ (5 mol. %). The mixture was stirred for 2-3 hours under reflux. After cooling, 100 ml CH₂Cl₂ was added and the organic phase was extracted with 10 % aqueous K₂CO₃ to remove the unreacted NDMBA. The organic layer was separated and the aqueous layer was extracted with CH₂Cl₂ (2×100 mL). The combined organic layers were dried over Na₂SO₄ and evaporated. The residue was purified by means of chromatography. Eluent: CH₂Cl₂/MeOH (50:1, 20:1, 10:1).

For some compounds, preparative HPLC followed by lyophilization was essential to obtain sufficient level of purity.

Purity of the final compounds was checked by LCMS (Column: Onyx C18 50x4.6mm | Solvent A : 0.1%TFA in AcN/H₂O (2.5:97.5), Solvent B : 0.1%TFA in AcN) 3.75mL/min; Gradient: "A"->2.2min->"B"(Hold 0.4min)->0.2min->"A"->Post Run).

4.1.15. 5-(4-chloro-3-fluorophenyl)-N-((5-(hydroxymethyl)-4-methylthiazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide

First fraction (21A ):

Yield: 40% (103 mg; over two steps). rt = 1.484 min. Purity = 96%. LC–MS: m/z [M⁺ +H] = 463 Da.

¹H NMR: (CDCl₃, 400 MHz) δ = 1.19 - 1.33 (m, 2 H), 1.36 - 1.78 (m, 6 H), 1.82 - 1.92 (m, 1 H), 2.30 (s, 3 H), 2.75 (t, J=11.7 Hz, 1 H), 3.15 - 3.29 (m, 1 H), 4.70 (s, 2 H), 5.35 - 5.46 (m, 1 H), 6.50 (d, J=3.7 Hz, 1 H), 6.95 (s, 1 H), 7.31 - 7.40 (m, 2 H), 7.43 (d, J=10.5 Hz, 1 H), 8.13 (br. s, 1 H), 10.67 (br. s, 1 H).

¹³C NMR (CDCl₃, 100 MHz): δ = 15.1, 24.0, 25.5, 29.8, 46.7, 54.2 (br.), 56.4, 59.1, 108.5, 112.9 (d, J = 22.0 Hz), 114.2, 119.5 (d, J = 18.3 Hz), 121.1, 126.9, 131.1, 132.1 (d, J = 8.8 Hz), 132.4 (d, J = 6.6 Hz), 134.1, 149.3, 158.5 (d, J = 248.8 Hz), 161.3, 168.6.

Second fraction (21B):

Yield: 49% (99 mg; over two steps). rt = 1.496 min. Purity = 93%. LC–MS: m/z [M⁺ +H] = 463 Da.

¹H NMR: (CDCl₃, 400 MHz) δ = 1.19 - 1.35 (m, 2H), 1.35 - 1.52 (m, 3H), 1.65 - 1.73 (m, 1 H), 1.74 - 1.92 (m, 2 H), 2.31 (s, 3 H), 2.72 (t, J=10.6 Hz, 1 H), 3.15 - 3.32 (m, 2 H), 4.71 (s, 2 H), 5.29 - 5.43 (m, 1 H), 6.48 (d, J=3.8 Hz, 1 H), 6.87 (d, J=3.1 Hz, 1 H), 7.31 - 7.38 (m, 2 H), 7.41 (d, J=10.5 Hz, 1 H), 8.04 (br. s, 1 H), 10.50 (br. s, 1 H).

4.1.16. 5-(3-fluoro-4-methylphenyl)-N-((5-(hydroxymethyl)-4-methylthiazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide

First fraction (22A):

Yield: 58% (183 mg; over two steps). rt = 1.468 min. Purity = 100%. LC–MS: m/z [M⁺ +H] = 443 Da.

¹H NMR: (CDCl₃, 400 MHz) δ = 1.27 (s, 1H), 1.32 - 1.74 (m, 6 H), 1.82 (d, J=11.4 Hz, 1 H), 2.29 (s, 3 H), 2.32 (s, 3 H), 2.73 (t, J=11.1 Hz, 1 H), 3.12 - 3.23 (m, 1 H), 3.44 - 3.51 (m, 1 H), 4.72 (s, 2 H), 5.41 (d, J=5.9 Hz, 1 H), 6.49 (d, J=3.5 Hz, 1 H), 6.94 (d, J=3.3 Hz, 1 H), 7.18 (t, J=7.9 Hz, 1 H), 7.20 - 7.32 (m, 2 H), 8.06 (br. s, 1 H), 10.56 (br. s, 1 H).

¹³C NMR (CDCl₃, 100 MHz): δ = 14.5 (d, J = 2.9 Hz), 15.0, 23.9, 24.8, 28.3, 46.6, 54.0, 56.1, 59.2, 107.6, 111.4 (d, J = 24.9 Hz), 113.7, 120.2 (d, J = 4.4 Hz), 123.8 (d, J = 17.6 Hz), 126.0, 131.3 (d, J = 7.3 Hz), 132.0 (d, J = 5.9 Hz), 132.4, 135.4, 149.2, 161.4, 161.7 (d, J = 244.4 Hz), 168.5.

Second fraction (22B):

Yield: 34% (122 mg; over two steps). rt = 1.573 min. Purity = 100%. LC–MS: m/z [M⁺ +H] = 443 Da.

¹H NMR: (CDCl₃, 400 MHz) δ = 1.23 - 1.44 (m, 4 H), 1.51 - 1.59 (m, 1 H), 1.66 - 1.81 (m, 2 H), 2.23 (d, J=0.6 Hz, 3 H), 2.26 (s, 3 H), 2.57 (t, J=11.3 Hz, 1 H), 3.07 (d, J=11.6 Hz, 1 H), 3.65 (br. s., 3 H), 4.65 (s, 2 H), 5.25 - 5.34 (m, 1 H), 6.42 (d, J=3.8 Hz, 1 H), 6.89 (d, J=3.8 Hz, 1 H), 7.11 (t, J=8.1 Hz, 1 H), 7.18 - 7.27 (m, 1 H), 7.91 (br. s, 1 H), 10.60 (br. s, 1 H).

¹³C NMR (CDCl₃, 100 MHz): δ = 14.3 (d, J = 2.9 Hz), 14.9, 24.0, 25.8, 28.8, 46.4, 53.9, 56.0, 60.2, 107.6, 111.2 (d, J = 24.2 Hz), 113.7, 120.0 (d, J = 2.9 Hz), 123.7 (d, J = 17.6 Hz), 126.0, 131.3 (d, J = 8.8 Hz), 131.9 (d, J = 5.9 Hz), 132.5, 135.3 (d, J = 2.2 Hz), 148.8, 161.0, 161.6 (d, J = 244.4 Hz), 166.6.

4.1.17. 5-(3-fluoro-4-methylphenyl)-N-((5-(2-hydroxyethyl)-4-methylthiazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide

First fraction (23A):

Yield: 17% (53 mg; over two steps). rt = 1.492 min. Purity = 100%. LC–MS: m/z [M⁺ +H] = 457 Da.

¹H NMR: (CDCl₃+CD₃OD, ~5:1, 400 MHz) δ = 1.17 (s, 1 H), 1.39 - 1.55 (m, 1 H), 1.63 - 1.91 (m, 5 H), 2.17 (s, 3 H), 2.24 (s, 3 H), 2.75 - 2.91 (m, 2 H), 3.37 (d, J=12.1 Hz, 1 H), 3.62 (t, J=6.2 Hz, 2 H), 3.66 - 3.72 (m, 1 H), 3.84 (br. s., 4 H), 5.54 (d, J=4.2 Hz, 1 H), 6.41 (d, J=3.7 Hz, 1 H), 7.03 - 7.13 (m, 2 H), 7.29 (dd, J=15.0, 8.5 Hz, 2 H).

¹³C NMR: (CDCl₃+CD₃OD, ~5:1, 100 MHz) δ = 14.1 (d, J= 3.7 Hz), 14.6, 22.5, 22.8, 26.5, 29.5, 45.8, 52.0, 59.3, 62.0, 107.5, 111.2 (d, J= 24.2 Hz), 115.7, 120.1 (d, J= 2.9 Hz), 123.7 (d, J= 17.6 Hz), 125.2, 130.2, 131.1 (d, J= 8.1 Hz), 131.8 (d, J= 5.9 Hz), 135.7 (d, J=2.2 Hz), 148.6, 161.4, 161.5 (d, J= 244.4 Hz), 165.0.

Second fraction (23B):

Yield: 9% (72 mg; over two steps). rt = 1.436 min. Purity = 98%. LC–MS: m/z [M⁺ +H] = 457 Da.

¹H NMR: (DMSO-d₆, 400 MHz) δ = 1.08 - 1.20 (m, 1 H), 1.28 (t, J=8.7 Hz, 2 H), 1.41 - 1.51 (m, 1 H), 1.73 (d, J=8.7 Hz, 2 H), 2.21 (s, 3 H), 2.25 (s, 3 H), 2.80 (t, J=6.4 Hz, 2 H), 2.91 - 3.03 (m, 2 H), 3.49 - 3.56 (m, 2 H), 4.81 (br. s., 1 H), 5.14 (t, J=8.6 Hz, 1 H), 6.61 (d, J=3.2 Hz, 1 H), 6.96 (d, J=3.3 Hz, 1 H), 7.24 (t, J=8.1 Hz, 1 H), 7.52 (dd, J=7.9, 1.2 Hz, 1 H), 7.62 (d, J=11.6 Hz, 1 H), 8.48 (d, J=8.9 Hz, 1 H), 11.75 (br. s, 1 H). Two exchangeable protons are missed.

¹³C NMR (DMSO-d₆, 100 MHz): δ = 13.9 (d, J = 3.2 Hz), 14.9, 24.1, 25.8, 28.8, 29.5, 46.1, 54.4, 59.0, 61.3, 107.4, 110.9 (d, J = 24.1 Hz), 113.1, 120.4 (d, J = 3.2 Hz), 122.3 (d, J = 16.9 Hz), 127.1, 128.5, 131.6 (d, J = 8.8 Hz), 131.8 (d, J = 5.6 Hz), 133.9 (d, J = 2.4 Hz), 147.3, 159.8, 161.0 (d, J = 241.7 Hz), 167.4.

HRMS (ESI): m/z calcd for C₂₄H₃₀FN₄O₂S [M+H]⁺ 457.2068, found 457.2071.

4.1.18. 5-(4-chloro-3-fluorophenyl)-N-((5-(2-hydroxyethyl)-4-methylthiazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide

First fraction (24A):

Yield: 26% (58 mg; over two steps). rt = 1.458 min. Purity = 97%. LC–MS: m/z [M⁺ +H] = 477 Da.

¹H NMR: (CDCl₃+CD₃OD, ~1:1, 400 MHz) δ = 1.09 (s, 1 H), 1.33 - 1.48 (m, 1 H), 1.55 - 1.84 (m, 5 H), 2.17 (s, 3 H), 2.74 (t, J=5.7 Hz, 2H), 2.77 - 2.88 (m, 1 H), 3.14 - 3.23 (m, 1 H), 3.31 (d, J=12.1 Hz, 1 H), 3.54 (t, J=6.2 Hz, 2 H), 3.59 - 3.67 (m, 1 H), 4.22 (br. s., 3 H), 5.45 (d, J=5.1 Hz, 1 H), 6.37 (d, J=3.9 Hz, 1 H), 6.96 (d, J=3.9 Hz, 1 H), 7.22 (t, J=7.9 Hz, 1 H), 7.27 - 7.34 (m, 1 H), 7.37 (d, J=10.4 Hz, 1 H).

¹³C NMR: (CDCl₃+CD₃OD, ~1:1, 100 MHz) δ = 14.3, 22.2, 22.5, 29.3, 45.6, 52.0, 59.0, 61.7, 108.1, 112.4, 112.6, 115.5, 119.0 (d, J = 18.3 Hz), 121.0 (d, J = 2.9 Hz), 125.8, 130.1, 130.7, 132.1 (d, J = 7.3 Hz), 134.4 (d, J = 2.2 Hz), 148.5, 158.1 (d, J = 247.4Hz), 161.2, 164.4.

Second fraction (24B):

Yield: 11% (109 mg; over two steps). rt = 1.468 min. Purity = 97%. LC–MS: m/z [M⁺ +H] = 477 Da.

¹H NMR: (CDCl₃, 400 MHz) δ = 1.18 - 1.52 (m, 5 H), 1.64 (d, J=9.8 Hz, 1 H), 1.70 - 1.89 (m, 2 H), 2.29 (s, 3 H), 2.65 (t, J=10.6 Hz, 1 H), 2.89 (t, J=5.3 Hz, 2 H), 3.16 (d, J=3.8 Hz, 2 H), 3.77 (t, J=5.6 Hz, 2 H), 5.22 - 5.47 (m, 1 H), 6.47 (d, J=3.4 Hz, 1 H), 6.83 (s, 1 H), 7.22 - 7.38 (m, 2 H), 7.41 (d, J=10.4 Hz, 1 H), 7.95 (br. s., 1 H), 10.76 (br. s., 1 H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 15.1, 24.0, 25.7, 28.7, 29.9, 46.4, 53.4, 60.6, 62.5, 108.5, 112.8 (d, J = 22.7 Hz), 113.4, 119.4 (d, J = 17.6 Hz), 121.1 (d, J = 2.9 Hz), 127.0, 129.1, 131.1, 132.4 (d, J = 8.1 Hz), 134.1 (d, J = 2.2 Hz), 148.8, 158.5 (d, J = 248.1 Hz), 160.7, 164.9.

HRMS (ESI): m/z calcd for C₂₃H₂₇ClFN₄O₂S [M+H]⁺ 477.1522, found 477.1522.

4.1.19. 5-(4-chlorophenyl)-N-((5-(2-hydroxyethyl)-4-methylthiazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide

First fraction (25A):

Yield: 24% (295 mg; over two steps). rt = 1.423 min. Purity = 95%. LC–MS: m/z [M⁺ +H] = 459 Da.

¹H NMR: (DMSO-d₆, 400 MHz) δ = 1.15 - 1.39 (m, 4 H), 1.47 - 1.57 (m, 2 H), 1.65 - 1.74 (m, 1 H), 2.24 (s, 3 H), 2.57 (t, J=9.9 Hz, 1 H), 2.80 (t, J=6.2 Hz, 2 H), 2.95 - 3.08 (m, 1 H), 3.13 - 3.22 (m, 1 H), 3.52 (t, J=6.0 Hz, 2 H), 4.81 (br. s., 1 H), 5.20 (t, J=7.5 Hz, 1 H), 6.62 (d, J=3.1 Hz, 1 H), 6.96 (d, J=3.1 Hz, 1 H), 7.41 (d, J=8.3 Hz, 2 H), 7.82 (d, J=8.3 Hz, 2 H), 8.51 (d, J=8.1 Hz, 1 H), 11.88 (br. s, 1 H)

¹³C NMR (DMSO-d₆, 100 MHz): δ = 14.9, 23.6, 25.1, 28.5, 29.5, 45.9, 54.5, 58.4, 61.3, 107.6, 113.5, 126.4 (2C), 127.3, 128.7 (3C), 130.7, 131.1, 133.8, 147.4, 160.1, 166.4.

HRMS (ESI): m/z calcd for C₂₃H₂₈ClN₄O₂S [M+H]⁺ 459.1616, found 459.1619.

Second fraction (25B):

Yield: 21% (141 mg; over two steps). rt = 1.500 min. Purity = 100%. LC–MS: m/z [M⁺ +H] = 459 Da.

¹H NMR: (DMSO-d₆, 400 MHz) δ = 1.13 - 1.39 (m, 4 H), 1.47 - 1.55 (m, 1 H), 1.70 - 1.80 (m, 2 H), 2.25 (s, 3 H), 2.56 (t, J=10.3 Hz, 1 H), 2.80 (t, J=6.2 Hz, 2 H), 3.00 (d, J=11.1 Hz, 1 H), 3.05 - 3.16 (m, 1 H), 3.48 - 3.56 (m, 2 H), 4.78 - 4.84 (m, 1 H), 5.21 (t, J=8.3 Hz, 1 H), 6.62 (d, J=2.8 Hz, 1 H), 6.98 (d, J=3.1 Hz, 1 H), 7.41 (d, J=7.8 Hz, 2 H), 7.82 (d, J=7.8 Hz, 2 H), 8.58 (d, J=8.9 Hz, 1 H), 11.86 (br. s, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz): δ = 14.9, 23.8, 25.2, 28.2, 29.5, 45.9, 54.0, 58.9, 61.3, 107.6, 113.3, 126.4 (2C), 127.3, 128.6 (2C), 128.7, 130.7, 131.1, 133.8, 147.4, 159.8, 167.1.

HRMS (ESI): m/z calcd for C₂₃H₂₈ClN₄O₂S [M+H]⁺ 459.1616, found 459.1624.

4.1.20. 5-(3-fluoro-4-methylphenyl)-N-((5-(3-hydroxypropyl)-4-methylthiazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide (26B)

Yield: 61% (790 mg; over two steps). rt = 1.491 min. Purity = 93%. LC–MS: m/z [M⁺ +H] = 471 Da.

¹H NMR: (CDCl₃, 400 MHz) δ = 1.17 - 1.41 (m, 4 H), 1.51 (d, J=10.5 Hz, 1 H), 1.61 - 1.81 (m, 4 H), 2.15 (s, 3 H), 2.21 (s, 3 H), 2.52 (t, J=11.3 Hz, 1 H), 2.67 (t, J=7.3 Hz, 2 H), 2.95 - 3.06 (m, 2 H), 3.60 (t, J=6.1 Hz, 2 H), 4.71 (br. s., 1 H), 5.29 (dd, J=7.9, 5.4 Hz, 1 H), 6.44 (d, J=3.5 Hz, 1 H), 6.86 (d, J=3.5 Hz, 1 H), 7.08 (t, J=8.0 Hz, 1 H), 7.21 - 7.26 (m, 2 H), 7.83 (d, J=7.1 Hz, 1 H), 10.86 (br. s., 1 H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 14.3 (d, J = 3.2 Hz), 14.7, 22.7, 24.4, 26.4, 29.4, 34.2, 46.5, 53.8, 60.5, 61.0, 107.5, 111.3 (d, J = 23.3 Hz), 112.8, 120.1 (d, J = 3.2 Hz), 123.4 (d, J = 17.7 Hz), 126.4, 131.5 (d, J = 8.0 Hz), 131.8 (d, J = 5.6 Hz), 131.9, 135.1 (d, J = 2.4 Hz), 147.7, 160.7, 161.5 (d, J = 244.1 Hz), 164.1.

HRMS (ESI): m/z calcd for C₂₅H₃₂FN₄O₂S [M+H]⁺ 471.2225, found 471.2225.

4.1.21. 5-(4-chloro-3-fluorophenyl)-N-((5-(3-hydroxypropyl)-4-methylthiazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide (27B)

Yield: 47% (640 mg; over two steps). rt = 1.518 min. Purity = 95%. LC–MS: m/z [M⁺ +H] = 491 Da.

¹H NMR: (CDCl₃, 400 MHz) δ = 0.84 - 0.96 (m, 1 H), 1.23 - 1.55 (m, 5 H), 1.59 - 1.72 (m, 1 H), 1.78 (ddd+m, J=14.0, 6.9, 6.7 Hz, 2+1 H), 1.84 - 1.92 (m, 1 H), 2.24 (s, 3 H), 2.73 (t, J=7.6 Hz, 2 H), 3.15 - 3.29 (m, 2 H), 3.63 (t, J=6.3 Hz, 1 H), 2.50 - 4.00 (br. s, 2H), 5.31 - 5.38 (m, 1 H), 6.51 (d, J=3.9 Hz, 1 H), 6.88 (d, J=3.8 Hz, 1 H), 7.31 - 7.42 (m, 1 H), 7.45 (d, J=10.8 Hz, 1 H), 8.03 (br. s., 1 H), 10.72 (br. s, 1 H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 14.9, 22.7, 24.1, 25.9, 28.9, 34.2, 46.4, 53.3, 60.7, 61.4, 108.3, 112.9 (d, J = 22.4 Hz), 113.4, 119.3 (d, J = 18.1 Hz), 121.1 (d, J = 3.5 Hz), 127.1, 131.1, 132.4, 132.5 (d, J = 7.8 Hz), 134.0 (d, J = 1.7 Hz), 147.7, 158.5 (d, J = 248.3 Hz), 160.7, 164.0.

HRMS (ESI): m/z calcd for C₂₄H₂₉ClFN₄O₂S [M+H]⁺ 491.1678, found 491.1680.

4.1.22. 5-(4-chlorophenyl)-N-((5-(3-hydroxypropyl)-4-methylthiazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide (28B)

Yield: 24% (105 mg; over two steps). rt = 1.473 min. Purity = 98%. LC–MS: m/z [M⁺ +H] = 473 Da.

¹H NMR: (CDCl₃, 400 MHz) δ = 1.43 - 1.75 (m, 2 H), 1.80 (quin, J = 6.9 Hz, 2 H), 1.87 - 2.05 (m, 4 H), 2.30 (s, 3 H), 2.77 (t, J = 7.5 Hz, 2 H), 2.95 (td, J = 12.9, 2.3 Hz, 1 H), 3.63 (t + br. s, J = 6.2 Hz, 2+2H), 5.73 (dd, J = 8.1, 2.8 Hz, 1 H), 6.50 (dd, J = 3.5, 2.3 Hz, 1 H), 7.04 (dd, J = 3.6, 2.3 Hz, 1 H), 7.32 (d, J = 8.6 Hz, 2 H), 7.29 - 7.35 (m, 2 H), 7.60 (d, J = 8.6 Hz, 2 H), 9.64 (d, J = 8.1 Hz, 1 H), 11.34 (br. s, 1 H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 14.9, 22.59, 22.64, 22.9, 26.4, 34.0, 45.2, 51.1, 61.0, 61.4, 107.7, 116.2, 126.0 (2C), 126.4, 129.1 (2C), 130.6, 132.8, 133.6, 135.4, 147.2, 161.0, 169.0.

HRMS (ESI): m/z calcd for C₂₄H₃₀ClN₄O₂S [M+H]⁺ 473.1773, found 473.1770.

4.1.23. (4-methyl-1H-imidazol-5-yl)methanol hydrochloride (29)

A mixture containing 4-methylimidzole (30.0 g, 365 mmol), paraformaldehyde (12.0 g, 402 mmol), K₂CO₃ (55.0 g, 402 mmol) and isopropanol (150 mL) was heated at 60 °C for two days (~16h). The reaction mixture was cooled to RT and filtered. The inorganic material was washed with isopropanol (50 mL). Concentrated aqueous HCl (79 mL) was added to the filtrate and the solvent was removed in vacuo. The residue was recrystallized from ethanol (~50 mL), filtered and washed two times with acetone: ethanol (1:1) mixture.

Yield: 27% (14.88 g).

¹H NMR: (DMSO-d₆, 400 MHz) δ = 2.26 (s, 3H), 4.46 (s, 2H), 8.94 (s, 1H), 14.58 (br. s., 2H).

¹³C NMR (DMSO-d₆, 100 MHz): δ = 8.6, 52.0, 125.2, 128.6, 132.1.

4.1.24. 5-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-1H-imidazole (30)

4-Hydroxymethyl-5-methylimidazole hydrochloride (14.88 g, 100.5 mmol) was dissolved in DMF (150 mL). TBSC1 (22.70 g, 150.6 mmol, 1.5 equiv) and imidazole (18.97 g, 279 mmol, 2.8 equiv) were added and the reaction mixture stirred at r.t. for 24 hours. The reaction mixture was heated until the point where a homogeneous solution was formed (60-80 °C) and kept at this temperature for two hours. The reaction mixture was cooled to r.t. poured into water and extracted with EtOAc (3×100 mL). The organic layer was washed with brine, dried over Na₂SO₄ and evaporated. The residue was purified by chromatography (eluent: 10:1, CH₂Cl₂/MeOH, R_f =0.35 in the same system). Yield: 74%( 6.83 g;Yellow solid).

¹H NMR: (CDCl₃, 400 MHz) δ = 0.06 (s, 6H), 0.89 (s, 9H), 2.24 (s, 3H), 4.67 (s, 2H), 7.49 (s, 1H), 10.28 (br. s, 1H).

¹³C NMR: (CDCl₃, 100 MHz) δ = -5.2 (2C), 10.9, 18.5, 26.1 (3C), 57.4, 128.0, 130.6, 133.3.

4.1.25. 5-(((tert-butyldimethylsilyl)oxy)methyl)-N,N,4-trimethyl-1H-imidazole-1-sulfonamide (31A) and 4-(((tert-butyldimethylsilyl)oxy)methyl)-N,N,5-trimethyl-1H-imidazole-1-sulfonamide (31B)

To a stirred solution of 5-(((tert-butyldimethylsilyl)oxy)methyl)-4-methyl-1H-imidazole (15.40 g, 68.1 mmol) in CH₂Cl₂ (140 mL) was added Et₃N (9.5 mL, 68.1 mmol) followed by Me₂NSO₂Cl (7.33 mL, 68.1 mmol). The reaction mixture was heated under reflex for 2 hours cooled and washed with water (100 mL). The organic layer was dried over Na₂SO₄ and evaporated.

Chromatographic purification (eluent 5:1→3:1→0:1, hexanes/EtOAc). First fraction = 4.76 g (R_f =0.7, 1:1, hexanes/EtOAc).

Second fraction = 6.50 g

Third fraction: Yield: 75% (5.72 g; R_f =0.55, 1:1, hexanes/EtOAc). Typically, a mixture of two spots was collected and used as such. First fraction (31A):

¹H NMR: (CDCl₃, 400 MHz) δ = 0.10 (s, 6H), 0.91 (s, 9H), 2.42 (s, 3H), 2.90 (s, 6H), 4.64 (s, 2H), 7.82 (s, 1H).

¹³C NMR: (CDCl₃, 100 MHz) δ = -5.1 (2C), 9.9, 18.5, 26.0 (3C), 38.1, 59.1, 124.8, 136.7, 139.6.

Third fraction (31B):

¹H NMR: (CDCl₃, 400 MHz) δ = 0.13 (s, 6H), 0.92 (s, 9H), 2.25 (s, 3H), 2.93 (s, 6H), 4.78 (s, 2H), 7.85 (s, 1H).

¹³C NMR: (CDCl₃, 100 MHz) δ = -5.2 (2C), 13.1, 18.6, 26.0 (3C), 38.2, 53.5, 126.1, 137.7, 139.5.

4.1.26. allyl 2-(amino(5-(hydroxymethyl)-4-methyl-1H-imidazol-2-yl)methyl)piperidine-1-carboxylate (33)

A mixture of protected imidazoles 31A and 31B (6.97 g, 20.9 mmol, 2.0 equiv) was dissolved in THF (21 mL) and cooled to -78 °C. At this temperature n-BuLi (2.5 M, 8.4 mL, 21 mmol, 2.0 equiv) was added dropwise under nitrogen. The reaction mixture was stirred for 10 minutes at - 78 °C, and imine 2 (3.14 g, 10.5 mmol) was added dropwise as a solution in THF (21 mL). The reaction mixture was slowly (~1 hour) warmed to 0 °C, and poured into saturated NH₄Cl (0.1 L). The biphasic mixture was extracted with CH₂Cl₂ (3×100 mL). The combined organic phases were dried over Na₂SO₄, and evaporated. The residue was dissolved in 1M HCl-MeOH solution (200 mL). After dissolution the reaction mixture was stirred for 1 hour, evaporated (no heating), dissolved in CH₂Cl₂ and washed with 10 % aqueous K₂CO₃. The organic layer was dried over Na₂SO₄, evaporated and loaded on silica. Chromatographic purification (eluent: 10:1, 4:1 CH₂Cl₂/MeOH; TLC in 1:1 CH₂Cl₂/MeOH) yielded two fractions (33A and 33B).

First fraction; Yield: 31% (1.013 g; over two steps).

Second fraction: Yield: 29% (0.930 g; over two steps). First fraction (33A):

¹H NMR: (CDCl₃, 400 MHz) δ = 1.13 - 1.32 (m, 2 H), 1.32 - 1.53 (m, 3 H), 1.54 - 1.70 (m, 2 H), 2.10 (s, 3 H), 2.82 - 2.97 (m, 1 H), 4.01 - 4.13 (m, 1 H), 4.47 (br. s + m, 2 + 1H), 4.52 - 4.63 (m, 2 H), 5.18 (d, J=10.4 Hz, 1 H), 5.27 (dd, J=17.0, 1.1 Hz, 1 H), 5.50 - 6.82 (br. s, 4H), 5.84 - 5.97 (m, 1 H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 10.6, 19.2, 25.3, 25.7, 39.9, 48.0, 54.8, 55.6, 66.4, 117.6, 128.3, 130.9, 132.9, 146.2, 156.4.

Second fraction (33B):

¹H NMR: (CDCl₃, 400 MHz) δ = 1.32 - 1.49 (m, 2 H), 1.50 - 1.71 (m, 4 H), 2.14 (s, 3 H), 2.97 (t, J=12.8 Hz, 1 H), 3.92 (d, J=12.5 Hz, 1 H), 4.25 - 4.65 (m, 2H), 4.45 (br. s, 4 H), 5.10 - 5.18 (m, 2 H), 5.40 - 6.50 (br. s, 4H), 5.75 - 5.86 (m, 1 H).

¹³C NMR: (CDCl₃, 100 MHz) δ = 10.1, 18.8, 25.2 (2C), 29.8, 40.4, 48.4, 55.0, 66.3, 117.3, 126.5, 131.4, 132.9, 147.3, 155.7.

Compounds with general structure labeled as 34 were obtained following general procedure for amide coupling using amines 33A and 33B and acids 17-19 as substrates. Eluent: 10/1, CH₂Cl₂/MeOH (R_f=0.4 10:1, CH₂Cl₂/MeOH).

4.1.27. General procedure for deprotection (imidazoles).

To solution containing alloc-protected compound (1 mmol) and N,N'-dimethylbarbituric acid (3 mmol) in MeOH (10 mL), PPh₃ (10 mol. %) was added under a nitrogen atmosphere followed by Pd(dba)₂ (5 mol. %). The mixture was stirred for 2-3 hours under reflux. After cooling, the reaction mixture was evaporated and the residue was dissolved in CH₂Cl₂ (50 - 100 ml). The organic phase was extracted twice with 5 % aqueous HCl (~10 mL). The combined aqueous layers were evaporated and purified by preparative HPLC.

4.1.28. 5-(4-chlorophenyl)-N-((5-(hydroxymethyl)-4-methyl-1H-imidazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide (35)

First fraction (35A):

Yield: 5% (40.1 mg; over two steps). rt = 1.271 min. Purity = 97%. LC–MS: m/z [M⁺ +H] = 428 Da.

¹H NMR: (DMSO-d₆, 400 MHz) δ = 1.06 - 1.44 (m, 4 H), 1.45 - 1.60 (m, 1 H), 1.61 - 1.81 (m, 1 H), 2.09 (s, 3 H), 2.82 - 3.16 (m, 4 H), 3.41 (br. s, 3 H), 4.31 (s, 2 H), 4.56 - 4.96 (m, 1 H), 5.05 (br. s., 1 H), 6.63 (br. s., 1 H), 6.90 (br. s., 1 H), 7.44 (br. s., 1 H), 7.81 (br. s., 1 H), 8.16 (br. s., 1 H), 11.82 (br. s., 1 H).

Second fraction (35B):

Yield: 7% (51.1 mg; over two steps). rt = 1.121 min. Purity = 96%. LC–MS: m/z [M⁺ +H] = 428 Da.

¹H NMR: (DMSO-d₆, 400 MHz) δ = 1.36 (d, J=9.5 Hz, 4 H), 1.56 - 1.65 (m, 1 H), 1.67 - 1.83 (m, 2 H), 2.10 (s, 3 H), 2.71 - 2.82 (m, 1 H), 3.16 (d, J=11.9 Hz, 1 H), 3.28 (t, J=7.1 Hz, 1 H), 4.30 (s, 2 H), 5.28 (t, J=8.0 Hz, 1 H), 6.63 (d, J=2.2 Hz, 1 H), 6.92 (d, J=2.8 Hz, 1 H), 7.42 (d, J=8.6 Hz, 2 H), 7.82 (d, J=8.6 Hz, 2 H), 8.32 (s, 2 H), 8.66 (d, J=9.2 Hz, 1 H), 11.97 (br. s, 1 H).

¹³C NMR (DMSO-d₆, 100 MHz): δ = 10.5, 22.6, 23.6, 26.2, 45.1, 48.8, 54.4, 58.8, 107.5, 113.6, 126.2 (2C), 127.3, 127.6, 128.7 (2C), 130.7, 131.1, 131.4, 133.5, 143.5, 159.9, 164.9.

HRMS (ESI): m/z calcd for C₂₂H₂₇ClN₅O₂ [M+H]⁺ 428.1848, found 428.1847.

4.1.29. 5-(4-chloro-3-fluorophenyl)-N-((5-(hydroxymethyl)-4-methyl-1H-imidazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide

First fraction (36A):

Yield: 6% (41.6 mg; over two steps).

¹H NMR: (DMSO-d₆, 400 MHz) δ = 1.14 - 1.42 (m, 3 H), 1.47 - 1.59 (m, 2 H), 1.67 - 1.79 (m, 1 H), 2.25 (s, 3 H), 2.53 - 2.62 (m, 1 H), 2.97 - 3.07 (m, 1 H), 3.15 - 3.23 (m, 1 H), 4.53 (s, 2 H), 5.21 (t, J=7.8 Hz, 1 H), 6.73 (d, J=3.8 Hz, 1 H), 6.98 (d, J=3.8 Hz, 1 H), 7.56 (t, J=8.2 Hz, 1 H), 7.67 (dd, J=8.5, 1.5 Hz, 1 H), 7.91 (dd, J=11.2, 1.7 Hz, 1 H), 8.24 (s, 1 H), 8.64 (d, J=8.4 Hz, 1 H), 11.94 (br. s., 1 H). Two exchangeable protons are missed.

¹³C NMR (DMSO-d₆, 100 MHz): δ = 14.9, 23.5, 24.9, 28.4, 45.8, 54.6, 55.1, 58.2, 108.5, 112.5 (d, J = 22.5 Hz), 113.3, 117.1 (d, J = 17.7 Hz), 121.7 (d, J = 3.2 Hz), 127.8, 130.8, 132.7, 132.9 (d, J = 8.0 Hz), 133.0, 146.8, 157.5 (d, J = 244.1 Hz), 160.1, 164.1, 167.4.

Second fraction (36B):

Yield: 6% (53.2 mg; over two steps). rt = 1.160 min. Purity = 95%. LC–MS: m/z [M⁺ +H] = 446 Da.

¹H NMR: (DMSO-d₆, 400 MHz) δ = 1.16 - 1.43 (m, 4 H), 1.44 - 1.59 (m, 2 H), 1.70 - 1.79 (m, 2 H), 2.26 (s, 3 H), 2.61 (t, J=10.6 Hz, 1 H), 3.03 (d, J=11.6 Hz, 1 H), 3.13 - 3.22 (m, 1 H), 4.54 (s, 2 H), 5.26 (t, J=8.2 Hz, 1 H), 6.73 (d, J=3.7 Hz, 1 H), 7.00 (d, J=3.8 Hz, 1 H), 7.55 (t, J=8.2 Hz, 1 H), 7.67 (dd, J=8.6, 1.3 Hz, 1 H), 7.92 (dd, J=11.2, 1.7 Hz, 1 H), 8.22 (s, 1 H), 8.68 (d, J=8.9 Hz, 1 H), 11.99 (br. s., 1 H).

¹³C NMR (DMSO-d₆, 100 MHz): δ = 14.9, 23.5, 24.8, 27.7, 45.8, 53.9, 55.0, 58.7, 108.6, 112.6 (d, J = 22.7 Hz), 113.1, 117.1 (d, J = 22.7 Hz), 121.8 (d, J = 2.9 Hz), 127.8, 130.8, 132.8, 132.9 (d, J = 7.3 Hz), 133.2, 146.8, 157.5 (d, J = 245.2 Hz), 159.9, 163.9, 167.8.

HRMS (ESI): m/z calcd for C₂₂H₂₆ClFN₅O₂ [M+H]⁺ 446.1754, found 446.1755.

4.1.30. 5-(3-fluoro-4-methylphenyl)-N-((5-(hydroxymethyl)-4-methyl-1H-imidazol-2-yl)(piperidin-2-yl)methyl)-1H-pyrrole-2-carboxamide

First fraction (37A):

Yield: 6% (51.5 mg; over two steps). rt = 1.102 min. Purity = 100%. LC–MS: m/z [M⁺ +H] = 426 Da.

¹H NMR: (CD₃OD, 400 MHz) δ = 1.54 - 1.62 (m, 2 H), 1.66 - 1.84 (m, 3 H), 1.88 - 1.95 (m, 2 H), 2.23 (s, 3 H), 2.27 (d, J=1.2 Hz, 3 H), 3.05 (td, J=12.9, 3.0 Hz, 1 H), 3.43 - 3.50 (m, 1 H), 3.66 (s, 1 H), 4.50 (s, 2 H), 5.39 (d, J=7.7 Hz, 1 H), 6.56 (d, J=3.9 Hz, 1 H), 6.96 (d, J=4.0 Hz, 1 H), 7.25 (t, J=8.2 Hz, 1 H), 7.34 - 7.41 (m, 2 H), 8.38 (s, 2 H). Two exchangeable protons are missed.

¹³C NMR (CD₃OD, 100 MHz): δ = 10.1, 14.4 (d, J = 3.7 Hz), 23.1, 23.5, 27.4, 46.8, 50.1, 56.3, 60.7, 108.8, 112.2 (d, J = 24.2 Hz), 115.4, 121.6 (d, J = 2.9 Hz), 125.0 (d, J = 17.6 Hz), 127.2, 129.3, 133.1 (d, J = 8.1 Hz), 133.2 (d, J = 5.9 Hz), 134.0, 137.1 (d, J = 2.9 Hz), 143.7, 163.1 (d, J = 243.0 Hz), 163.5, 168.3.

δ = 49.15 ppm was used as a reference for methanol.

HRMS (ESI): m/z calcd for C₂₃H₂₉FN₅O₂ [M+H]⁺ 426.2300, found 426.2301.

Second fraction (37B):

Yield: 6% (54.5 mg; over two steps). rt = 1.115 min. Purity = 92%. LC–MS: m/z [M⁺ +H] =426 Da.

¹H NMR: (CD₃OD, 400 MHz) δ = 1.52 - 1.75 (m, 3 H), 1.87 - 1.99 (m, 2 H), 2.03 - 2.13 (m, 1 H), 2.23 (s, 3 H), 2.27 (s, 3 H), 3.10 (td, J=12.4, 2.6 Hz, 1 H), 3.44 - 3.51 (m, 1 H), 3.69 - 3.76 (m, 1 H), 4.50 (d, J=2.0 Hz, 2 H), 5.48 (d, J=6.8 Hz, 1 H), 6.57 (d, J=4.0 Hz, 1 H), 6.98 (d, J=3.9 Hz, 1 H), 7.26 (t, J=8.0 Hz, 1 H), 7.35 - 7.42 (m, 2 H), 8.34 (s, 2 H). Three exchangeable protons are missed.

¹³C NMR (CD₃OD, 100 MHz): δ = 9.9, 14.4 (d, J = 4.74 Hz), 23.0, 23.6, 26.4, 46.5, 49.2, 56.4, 60.6, 108.8, 112.2 (d, J = 23.4 Hz),115.3, 121.6 (d, J = 2.9 Hz), 125.0 (d, J = 17.6 Hz), 127.2, 129.0, 133.0 (d, J = 8.8 Hz), 133.2 (d, J = 4.4 Hz), 134.4, 144.0, 163.1 (d, J = 3.7 Hz), 163.4, 168.0.

HRMS (ESI): m/z calcd for C₂₃H₂₉FN₅O₂ [M+H]⁺ 426.2300, found 426.2303.

4.1.31. allyl 2,2-dimethoxyethylcarbamate (38)

Amino acetaldehyde dimethyl acetal (5.64 mL, 51.8 mmol, 1.10 equiv) and Et₃N (6.56 mL, 47.0 mmol) were dissolved in CH₂Cl₂ (94 mL) and alloc-Cl (5.00 mL, 47.0 mmol) was added dropwise with cooling of the flask on an ice bath. The reaction was stirred for 1 hour at r.t. and washed with 5-10 % aqueous citric acid. The aqueous layer was extracted with CH₂Cl₂ (100 mL). The combined organic layers were dried over Na₂SO₄ and evaporated.

Yield: 100% (8.86 g;Clear oil); Rf=0.6 (hexanes/EtOAc, 1:1).

¹H NMR (CDCl₃, 400 MHz): δ = 3.30 (t, J = 5.7 Hz, 2H), 3.37 (s, 6H), 4.36 (t, J = 5.3 Hz, 1H), 4.54 (d, J = 5.3 Hz, 2H), 5.07 (br. s, 1H), 5.19 (dd, J = 10.4, 1.2 Hz, 1H), 5.28 (dd, J = 17.2, 1.5 Hz, 1H), 5.90 (dddd, J = 16.8, 10.9, 5.6, 5.4 Hz, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = 42.5, 54.3 (2C), 65.7, 102.9, 117.7, 132.9, 156.4.

4.1.32. allyl allyl(2,2-dimethoxyethyl)carbamate (39)

Allyl 2,2-dimethoxyethylcarbamate (8.86 g, 46.9 mmol) was dissolved in DMF (47 mL) and NaH (60% dispersion in mineral oil, 2.10 g, 52.5 mmol, 1.10 equiv) was added in several portions. When the hydrogen evolution stops allyl bromide (4.47 mL, 51.7 mmol, 1.10 equiv) was added dropwise. After 10 minutes, the TLC shows disappearance of the starting material. The reaction mixture was diluted with water (100 mL) and extracted with EtOAc (3×100 mL). The combined organic layers were dried over Na₂SO₄ and evaporated. Yield: 100% (12.10 g; Clear oil); Rf=0.7 (hexanes/EtOAc, 3:1). Mixture of rotamers:

¹H NMR (CDCl₃, 400 MHz): δ = 3.25 - 3.33 (m, 2H), 3.36 (br. s, 6H), 3.89 - 4.00 (m, 2H), 4.46 (d, J = 18.8 Hz, 1H), 4.58 (d, J = 3.5 Hz, 2H), 5.04 - 5.15 (m, 2H), 5.17 (d, J = 10.4 Hz, 1H), 5.27 (d, J = 17.1 Hz, 1H), 5.67 - 5.81 (m, 1H), 5.84 - 5.97 (m, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = 48.1, 48.8, (50.6, 50.7), (54.53, 54.59), 66.1, 103.5, 103.9, 116.4, 116.9, 117.2, 117.4, 133.0, 133.5, 133.6, (155.8, 156.2).

4.1.33. allyl allyl(2-oxoethyl)carbamate (40)

Allyl allyl(2,2-dimethoxyethyl)carbamate was dissolved in formic acid (120 mL) and water (24 mL) was added to the solution. After 30 minutes the TLC shows disappearance of the starting material. After 1 day most of HCOOH was evaporated, the residue was dissolved in EtOAc (250 mL) and was washed with brine until neutral pH (10×100 mL). The organic layer was dried over Na₂SO₄ and evaporated to give pure enough aldehyde. Yield: 98% ( 8.37 g; over two steps). Yellow oil.

¹H NMR (CDCl₃, 400 MHz): δ = 3.97 (br. s, 2H), 3.98 - 4.05 (m, 2H), 4.61 (dd, J = 15.5, 5.0 Hz, 2H), 5.10 - 5.25 (m, 3H), 5.28(dd, J = 26.9, 17.2 Hz, 1H), 5.77 (ddt, J = 16.8, 10.5, 6.0 Hz, 1H), 5.91 (dddd, , J = 28.2, 22.6, 10.8, 5.5 Hz, 1H), 9.58 (s, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = 50.9, 51.3, 56.1, 56.6, 66.6, 117.8, 117.94, 117.98, 118.6, 132.5, 132.6, 133.0, 198.3.

4.1.34. (E)-allyl allyl(2-(tert-butylsulfinylimino)ethyl)carbamate (41)

To a solution of aldehyde (4.00 g, 21.9 mmol) in CH₂Cl₂ (22 mL) was added commercially available (S)-N-tert-butylsulfinylamide (2.91 g, 24.0 mmol, 1.10 equiv), PPTS (0.27 g, 0.050 equiv) and MgSO₄ (13.10 g, 109.2 mmol, 5.000 equiv). To control the progress of the reaction 0.5 mL of the DCM solution from the reaction mixture was evaporated and analyzed by NMR (once a day).The mixture was stirred at rt for 30 h, inorganic material was filtered and washed several times with CH₂Cl₂. The filtrate was concentrated in vacuo to give pure enough title compound (S)-41. Chromatographic purification: Hexanes/EtOAc 3:1. The product always contains inseparable mixture of aldehyde.

Yield: 91% (5.67 g). Clear oil.

The reaction with (R)-N-tert-butylsulfinylamide was conducted in the same manner affording the corresponding R enantiomer (R)-41. Yield: 76% (5.38 g).

¹H NMR (CDCl₃, 400 MHz): δ = 1.20 (s, 9H), 3.87 - 4.10 (m, 2H), 4.18 - 4.37 (m, 2H), 4.51 - 4.68 (m, 2H), 5.12 - 5.24 (m, 3H), 5.24 - 5.36 (m, 1H), 5.74 - 6.00 (m, 2H), 7.97 (t, J = 2.7 Hz, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = 22.3 (3C), 50.3, (50.6, 50.9), (57.0, 57.1), (66.3, 66.5), 117.5, (117.9, 118.1), (132.5, 132.7), 133.1, (155.6, 155.9), 165.1.

4.1.35. allyl allyl(2-(5-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylthiazol-2-yl)-2-(1,1-dimethylethylsulfinamido)ethyl)carbamate (42)

5-(((tert-butyldimethylsilyl)oxy)methyl)-4-methylthiazole (6.25 g, 25.7 mmol, 1.3 equiv) was dissolved in THF (25 mL) and cooled to -78 °C. At this temperature n-BuLi (2.5 M, 11 mL, 27.5 mmol, 1.4 equiv) was added dropwise under a nitrogen atmosphere. The reaction mixture was stirred for 20 minutes at -78 °C, and (S)-41 (5.67 g, 19.8 mmol) was added dropwise as a solution in THF (20 mL). The reaction mixture was slowly (~1 hour) warmed to 0 °C, and poured into water (0.1 L). The biphasic mixture was extracted with CH₂Cl₂ (3×100 mL). The combined organic phases were dried over Na₂SO₄, and evaporated to give brown oil which was purified by means of column chromatography. Eluent: hexanes/EtOAc (10:1, 5:1, then 1:1).

Yellow oil (fS-42): Rf = 0.4 (hexanes/EtOAc, 1:1). Yield: 66% (6.93 g).

The reaction with (R)-41 was conducted in the same manner to give fR-42.

Yield: 74% (7.40 g).

¹H NMR (CDCl₃, 400 MHz): δ = 0.09 (s, 6H), 0.90 (s, 9H), 1.27 (s, 9H), 2.32 (s, 3H), 3.55 (dd, J = 14.7, 2.5 Hz, 1H), 3.83 (dd, J = 16.4, 5.1 Hz, 1H), 3.88 - 4.03 (m, 2H), 4.54 - 4.69 (m, 2H), 4.76 (s, 2H), 4.89 (dt, J = 9.5, 3.8 Hz, 1H), 5.16 (dt, J = 15.9, 1.5 Hz, 3H), 5.19 (d, J = 17.1 Hz, 2H), 5.30 (dd, J = 17.2, 1.0 Hz, 1H), 5.56 (d, J = 2.4 Hz, 1H), 5.81 (ddd, J = 22.8, 10.6, 5.5, 1H), 5.93 (ddd, J = 22.5, 10.7, 5.4, 2H).

¹³C NMR (CDCl₃, 100 MHz): δ = -5.21 (2C), 15.3, 18.4, 22.9 (3C), 25.9 (3C), 29.8, 50.5, 52.3, 56.3, 58.0, 66.8, 117.0, 117.5, 132.8, 133.1, 133.2, 147.9, 158.6, 170.4.

4.1.36. allyl allyl(2-amino-2-(5-(hydroxymethyl)-4-methylthiazol-2-yl)ethyl)carbamate (43)

The 1M HCl-MeOH solution was prepared by dropwise addition of AcCl to a MeOH (100 mmol). The resulting solution was cooled to an ambient temperature and added to a flask containing 42 (10 mmol). After dissolution the reaction mixture was stirred for 1 hour, evaporated (no heating), dissolved in CH₂Cl₂ and washed with 10 % aqueous K₂CO₃. The organic layer was dried over Na₂SO₄, evaporated and loaded on silica. Eluting with CH₂Cl₂/MeOH (50:1) provided pure amine. Rf = 0.3 (CHCl₃/MeOH, 7:1). Yellow oil.

For fR-43: Yield: 75% (3.28 g).

For fS-43: Yield: 64% (2.62 g).

¹H NMR (CDCl₃, 400 MHz): δ = 2.30 (s, 3H), 2.62 (br. s., 3H), 3.45 - 3.68 (m, 2H), 3.74 - 3.94 (m, 2H), 4.36 (t, J = 6.7 Hz, 1H), 4.56 (d, J = 4.4 Hz, 2H), 4.69 (s, 2H), 5.12 (dd, J = 17.7, 9.5 Hz, 2H), 5.18 (d, J = 10.4 Hz, 1H), 5.28 (d, J = 17.2 Hz, 1H), 5.74 (ddt, J = 16.4, 10.7, 5.3 Hz, 1H), 5.89 (ddt, J = 16.7, 11.0, 5.3, 1H)

¹³C NMR (CDCl₃, 100 MHz): δ = 15.0, (50.6, 50.8), 53.1, (53.4, 54.1), 56.3, 66.4, 117.0, 117.5, 117.9, 131.7, 132.8, 133.2, 148.8, (156.2, 157.0), (171.7, 172.2).

4.1.37. allyl allyl(2-(5-(4-chlorophenyl)-1H-pyrrole-2-carboxamido)-2-(5-(hydroxymethyl)-4-methylthiazol-2-yl)ethyl)carbamate (44)

To a suspension/solution of 5-(4-chlorophenyl)-1H-pyrrole-2-carboxylic acid (878 mg, 3.96 mmol) in DMF (8.8 mL) DIPEA (0.690 mL, 3.97 mmol) was added followed by HBTU (1.506 g, 3.97 mmol). The resulting solution was stirred for 10 minutes and added to a solution of amine fS-43 (1.237 g, 3.97 mmol) in DMF (12 mL). The reaction mixture was stirred for 12 hours, evaporated, diluted with CH₂Cl₂ (100 mL) and successively washed with 5% aqueous NaOH and 10 % citric acid solutions. The organic layer was dried over Na₂SO₄, evaporated and loaded on silica. Eluting with hexanes: EtOAc (1:1, than pure EtOAc) gave the target compounds.

First enantiomer (fS-44): Yield: 74% (1.52 g).

The second enantiomer (fR-44) was prepared from amine fR-43 using the same procedure: Yield: 58% (1.11 g). Light brown oil.

¹H NMR (CDCl₃, 400 MHz): δ = 2.28 (s, 3H), 3.53 (dd, J = 14.5, 3.1 Hz, 1H), 3.72 (dd, J = 16.4, 5.6 Hz, 1H), 3.85 (dd, J = 16.2, 4.8 Hz, 1H), 4.07 (dd, J = 14.8, 10.8 Hz, 1H), 4.51 (d, J = 5.0 Hz, 2H), 4.18 - 4.95 (br. s, 1H), 4.65 (s, 2H), 5.05 - 5.22 (m, 4H), 5.38 (ddd, J = 9.8, 6.8, 3.4 Hz, 1H), 5.62 - 5.83 (m, 2H), 6.44 (t, J = 3.1 Hz, 1H), 6.80 (t, J = 2.3 Hz, 1H), 7.21 (d, J = 8.6 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 8.21 (d, J = 6.7 Hz, 1H), 10.76 (br. s, 1H).

¹³C NMR (CDCl₃, 100 MHz): δ = 14.8, 50.3, 50.6, 52.4, 56.0, 107.9, 113.0, 117.2, 117.7, 126.2 (2C), 126.4, 128.9 (2C), 130.4, 131.9, 132.2, 132.7, 132.8, 135.3, 149.1, 158.1, 161.6, 168.8.

4.1.38. N-(2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)ethyl)-5-(4-chlorophenyl)-1H-pyrrole-2-carboxamide (45)

To a solution containing fS-44 (R₁=H, R₂=Cl) (1.52 g, 2.95 mmol) and N,N'-dimethylbarbituric acid (2.30 g, 14.7 mmol, 5 equiv) in MeOH (30 mL), PPh₃ (77 mg, 20 mol. %) was added under a nitrogen atmosphere followed by Pd(dba)₂ (85 mg, 10 mol. %). The mixture was stirred for 6 hours at reflux. After cooling, 200 mL CH₂Cl₂ was added and the organic phase was extracted twice with 5 % aqueous K₂CO₃ to remove the unreacted NDMBA. The organic phase was dried over Na₂SO₄ and concentrated. Purification by flash chromatography (CH₂Cl₂: MeOH, 20:1, 5:1, 1:1) afforded amine 45A as a slightly brown or yellowish solids. Rf=0.5 (DCM/MeOH, 1:1).

Yield: 39% (450 mg); rt = 1.430 min. Purity = 100%. LC–MS: m/z [M⁺ +H] = 391 Da.

HRMS (ESI): m/z calcd for C₁₈H₂₀ClN₄O₂S [M+H]⁺ 391.0990, found 391.0992.

The second enantiomer 45B was prepared from fR-44 using the same procedure: Yield: 65% (550 mg); rt = 1.342 min. Purity = 100%. LC–MS: m/z [M⁺ +H] = 391 Da. HRMS (ESI): m/z calcd for C₁₈H₂₀ClN₄O₂S [M+H]⁺ 391.0990, found 391.0992.

¹H NMR (DMSO-d₆, 400 MHz): δ = 2.27 (s, 3H), 2.99 - 3.08 (m, J = 13.3, 7.9, 1H), 3.15 (dd, J = 13.1, 5.3 Hz, 1H), 4.54 (s, 2H), 5.19 (td, J = 7.8, 5.8 Hz, 1H), 6.65 (d, J = 3.9 Hz, 1H), 7.00 (d, J = 3.9 Hz, 1H), 7.43 (d, J = 8.7 Hz, 2H), 7.85 (d, J = 8.7 Hz, 2H), 8.57 (d, J = 8.1 Hz, 1H), 11.90 (br. s, 1H).

¹³C NMR (DMSO-d₆, 100 MHz): δ = 14.9, 45.4, 53.9, 55.1, 107.6, 113.1, 126.4 (2C), 127.5, 128.6 (2C), 130.7, 131.1, 132.6, 133.8, 146.9, 160.4, 169.2.

The enantiopurity of compounds 45A and 45B was checked on a chiral column ODRH (a mechanical mixture of enantiomers was used as a reference).

ee (45A) = 77% and ee (45B) = 88%.

Compounds 46A - 47B were prepared using the same two-step procedures as for compound 45A. Protected compounds were used (fR-44 and fS-44) in the next step after chromatographic purification without analysis.

4.1.39. N-(2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)ethyl)-5-(4-chloro-3-fluorophenyl)-1H-pyrrole-2-carboxamide [Two isomers, 46A and 46B

46A: Yield: 50% (755 mg; over two steps). rt = 1.275 min. Purity = 98%. LC–MS: m/z [M⁺ +H] =409 Da.

HRMS (ESI): m/z calcd for C₁₈H₁₉ClFN₄O₂S [M+H]⁺ 409.0896, found 409.0903.

46B: Yield: 54% (710 mg; over two steps). rt = 1.375 min. Purity = 97%. LC–MS: m/z [M⁺ +H] =409 Da.

HRMS (ESI): m/z calcd for C₁₈H₁₉ClFN₄O₂S [M+H]⁺ 409.0896, found 409.0900.

¹H NMR: (DMSO-d₆, 400 MHz) δ = 2.27 (s, 3H), 3.18 (dd, J = 13.0, 8.7 Hz, 1H), 3.30 (dd, J = 13.0, 5.1 Hz, 1H), 4.54 (s, 2H), 5.34 (td, J = 8.3, 5.1 Hz, 1H), 4.0 - 6.0 (br. s, 2H), 6.76 (d, J = 3.8 Hz, 1H), 6.99 (d, J = 3.9 Hz, 1H), 7.57 (t, J = 8.3 Hz, 1H), 7.70 (dd, J = 8.5, 1.8 Hz, 1H), 7.95 (dd, J = 11.2, 2.0 Hz, 1H), 8.97 (d, J = 8.1 Hz, 1H), 11.93 (br. s, 1H).

¹³C NMR (DMSO-d₆, 100 MHz): δ = 14.8, 43.4, 51.7, 55.0, 108.6, 112.6 (d, J = 22.7 Hz), 113.1, 117.0 (d, J = 17.6 Hz), 121.7 (d, J = 3.7 Hz), 127.9, 130.8, 133.7, 132.9 (d, J = 7.3 Hz), 133.1, 146.9, 157.5 (d, J = 245.2 Hz), 160.3, 168.0.

4.1.40. N-(2-amino-1-(5-(hydroxymethyl)-4-methylthiazol-2-yl)ethyl)-5-(3-fluoro-4-methylphenyl)-1H-pyrrole-2-carboxamide (Two isomers, 47Aand 47B)

47A (NBD-14012): Yield: 22% (313 mg; over two steps). rt = 1.316 min. Purity = 100%.

HRMS (ESI): m/z calcd for C₁₉H₂₂FN₄O₂S [M+H]⁺ 389.1442, found 389.1448.

47B: Yield: 37% (490 mg; over two steps). rt = 1.339 min. Purity = 100%.

HRMS (ESI): m/z calcd for C₁₉H₂₂FN₄O₂S [M+H]⁺ 389.1442, found 389.1445.

¹H NMR: (DMSO-d₆, 400 MHz) δ = 2.23 (s, 3H), 2.27 (s, 3H), 3.18 (dd, J = 13.1, 8.8 Hz, 1H), 3.31 (dd, J = 13.1, 5.0 Hz, 1H), 4.54 (s, 2H), 5.35 (td, J = 8.3, 5.1 Hz, 1H), 4.0-6.0 (br. s, 2H), 6.65 (d, J = 3.8 Hz, 1H), 6.96 (d, J = 3.8 Hz, 1H), 7.27 (t, J = 8.2 Hz, 1H), 7.56 (dd, J = 7.9, 1.7 Hz, 1H), 7.66 (dd, J = 11.6, 1.7 Hz, 1H), 8.93 (d, J = 7.9 Hz, 1H), 11.93 (br. s, 1H).

¹³C NMR (DMSO-d₆, 100 MHz): δ = 13.9 (d, J = 2.9 Hz), 14.8, 45.1, 53.4, 55.0, 107.4, 111.4 (d, J = 24.2 Hz), 112.9, 120.5, 122.3 (d, J = 17.6 Hz), 127.2, 131.6 (d, J = 8.1 Hz), 131.8 (d, J = 5.9 Hz), 132.7, 133.9 (d, J = 2.9 Hz), 146.9, 160.4, 161.0 (d, J = 242.2 Hz), 168.1.

4.2. Biology

4.2.1. Cells and viruses

MT-2 cells (Human T-cell leukemia cells) were obtained through the NIH AIDS Research and Reference Reagent Program (ARP) from Dr. D. Richman ²⁵. TZM-bl cells (a HeLa cell line that expresses CD4, CXCR4 and CCR5 and expresses luciferase and ß-galactosidase under control of the HIV-1 promoter) were obtained from Dr. J. C. Kappes, Dr. X. Wu and Tranzyme Inc. through the NIH ARP ²⁶. H9/HTLV-/HIV-1_IIIB cells were obtained through the NIH ARP from Dr. R. Gallo ²⁷. HEK 293T cells were purchased from ATCC. MOLT-4/CCR5 cells were obtained through the NIH ARP from Dr. M. Baba, Dr. H. Miyake and Dr. Y. Iizawa. GHOST X4/R5 cells were obtained through the NIH ARP from Drs. V. N. Kewalramani and D. R. Littman ²⁸. MAGI-CCR5 cells were obtained through the NIH ARP from Dr. J. Overbaugh ²⁹. HL2/3 cells were obtained through the NIH ARP from Drs. B. K. Felber and G. N. Pavlakis ³⁰. CD4-negative Cf2Th-CCR5+ cells and Env expression vector pSVIIIenv-ADA were kindly provided by Dr. J. G. Sodroski ³¹. HIV-1 Env molecular clone expression vector pHXB2-env (X4) DNA was obtained through the ARP from Dr. K. Page and Dr. D. Littman ³². 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 panel C (QB099.391M.ENV.B1 and QB099.391M.ENV.C8) were obtained through the NIH ARP from Dr. J. Overbaugh ^33;34. The HIV-1 Env molecular clones panel of subtype A/G and A/E (CRF01_AE clone 269) Env clones were obtained through the NIH ARP from Drs. D. Ellenberger, B. Li, M. Callahan and S. Butera ³⁵. The AE clone AA058 was kindly provided by Drs. R. J. McLinden and A. L. Chenine from US Military HIV Program, Henry M. Jackson Foundation (Silver Spring, MD).The HIV-1 Env panel of standard reference subtype B Env clones were obtained through the NIH ARP from Drs. D. Montefiori, F. Gao and M. Li (PVO, clone 4 (SVPB11); TRO, Clone 11 (SVPB12); AC10.0, clone 29 (SVPB13); QH0692, clone 42 (SVPB6); SC422661, clone B (SVPB8)); from Drs. B. H. Hahn and J. F. Salazar-Gonzalez (pREJO4541, clone 67 (SVPB16); pRHPA4259, clone 7 (SVPB14); pWITO4160 clone 33 (SVPB18)); from Drs. B. H. Hahn and D. L. Kothe (pTHRO4156 clone 18 (SVPB15), pCAAN5342 clone A2 (SVPB19)) ^26;36;37. The subtype B pWEAUd15.410.5017 and p1058_11.B11.1550 were obtained through the NIH ARP from Drs. B. H. Hahn, B. F. Keele and G. M. Shaw ³⁸. The subtype C HIV-1 reference panel of Env clones were also obtained through the NIH ARP from Drs. D. Montefiori, F. Gao, S. A. Karim and G. Ramjee (Du 156.12; 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; ZM233M.PB6; ZM214M.PL15); from Drs. E. Hunter and C. Derdeyn (ZM53M.PB12; ZM135M.PL10a; ZM109F.PB4); from Drs. L. Morris, K. Mlisana and D. Montefiori, (CAP45.2.00.G3; CAP210.2.00.E8) ^39-41. The HIV-1 Subtype C Panel of Indian gp160 Env Clone HIV-16055-2, clone 3 was obtained through the NIH ARP from Drs. R. Paranjape, S. Kulkarni and D. Montefiori ³⁵. HIV-1 Env molecular clones MF535.W0M.ENV.C1 and BF535.W6M.ENV.A1 of subtype D/A, were obtained through the NIH ARP from Dr. J. Overbaugh ⁴². The ENV pseudotyped genes of BG505.T332N, KNH1144 and B41 were kindly provided by Dr. J. P. Moore of the Weil Cornell Medical College, NY.

The Env-deleted pro-viral backbone plasmids, pNL4-3.Luc.R-.E-DNA (Dr. N. Landau) ^43;44 and pSG3Δ^env DNA (Drs. J. C. Kappes and X. Wu) ^26;37 were obtained through the NIH ARP. MLV gag-pol-expressing vector pVPack-GP, Env-expressing vector pVPack-VSV-G and a pFB-luc vector were obtained from Stratagene (La Jolla, CA). HIV-1_IIIB laboratory adapted strain was obtained through the NIH ARP.

4.2.2. Pseudovirus preparation

Pseudoviruses capable of single cycle infection were prepared as previously described¹⁸. Briefly, HEK 293T cells were transfected with a mixture of 10 μg of an env-deleted pro-viral backbone plasmid pNL4-3.Luc.R-E-DNA or pSG3^Δenv DNA and 10 μg of an HIV-1 Env expression vector using FuGENE 6 (Roche). VSV-G pseudovirus was prepared by transfecting 293T cell with a mixture of 10 μg each of the Env-expressing vector pVPack-VSV-G, the MLV gag-pol-expressing vector pVPack-GP and the pFB-luc vector using FuGENE 6. Pseudovirus-containing supernatants were collected two days after transfection, filtered, and stored in aliquots at −80 °C. Pseudoviruses were tittered to calculate the 50% tissue culture infectious dose (TCID₅₀) by infecting different cell types.

4.2.3. Measurement of antiviral activity

Single-cycle infection assay in TZM-bl cells

The inhibitory activity of the new generation of NBD molecules was tested against the pseudovirus HIV-1_HXB-2 (X4) in a single cycle infection assay. Additionally, selected small molecules 45A and 46A were also tested against a large group of HIV-1 pseudotyped viruses expressing the Env from the panel of standard reference as previously described¹⁸. Briefly, TZM-bl cells were plated at 10⁴ cells / well in a 96-well tissue culture plate and cultured at 37 °C overnight. Graded concentrations of the small molecules were incubated with HIV-1 pseudovirus for 30 minutes. The mixture was added to the cells and cultured for 3 days. Cells were washed with PBS and lysed with 50 μl of cell culture lysis reagent (Promega). 20 μl of lysates were transferred to a white plate and mixed with 100 μl of luciferase assay reagent (Promega). The luciferase activity was immediately measured with a Tecan infinite M1000 reader and the percent inhibition by the compounds and IC₅₀ values were calculated using the GraphPad Prism software (GraphPad).

Multi-cycle infection assay

We evaluated the inhibitory activity of the small molecules on infection by the laboratory-adapted HIV-1_IIIB as previously described ⁴⁵. Briefly, 10⁴ MT-2 cells / well were infected with HIV-1_IIIB at 100 TCID₅₀ (0.0069 MOI) pre-treated for 30 minutes with graded concentrations of small molecules. Following overnight incubation at 37 ºC, the culture supernatants were replaced with fresh media. Four days post-infection, the culture supernatants were collected and mixed with an equal volume of 5 % Triton X-100 and tested for p24 antigen by sandwich-ELISA. The percent inhibition of p24 production and IC₅₀ (the half maximal inhibitory concentration) values were calculated by the GraphPad Prism software.

4.2.4. Evaluation of cytotoxicity

TZM-bl cells

The cytotoxicity of the small molecules in TZM-bl cells was measured by the XTT method. Briefly, 100 μl of a compound at graded concentrations was added to equal volume of cells (10⁵/ml) in wells of 96-well plates followed by incubation for 3 days and addition of XTT as described above. The percent of cytotoxicity and the CC₅₀ values were calculated as above.

MT-2 cells

The cytotoxicity of the small molecules in MT-2 cells which ran parallel to the antiviral assay was measured with a colorimetrical method using XTT (PolySciences) as previously described⁴⁵. Briefly, 100 μl of a small molecule at graded concentrations was added to an equal volume of cells (10⁵ cells / ml) in 96-well plates followed by incubation for 4 days. Four hours after the addition of XTT the soluble intracellular formazan was quantitated at 450 nm. The percent of cytotoxicity and the CC₅₀ (the concentration for 50 % cytotoxicity) values were calculated as above.

4.2.5. 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 overnight. The cells were infected with the luciferase expressing recombinant CD4-dependent pseudovirus HIV-1_ADA as previously described ¹⁰. Briefly, 50 μl of a test compound at graded concentrations was mixed with equal volume of the recombinant virus and incubated for 30 minutes. The mixtures were added to the cells and cultured for 48 hours. Cells were washed with PBS and lysed with 40 μl of cell lysis reagent. Lysates were transferred to a white 96-well plate and mixed with 100 μl of luciferase assay reagent. The luciferase activity was immediately measured to obtain the relative infection with respect to the untreated control.

4.2.6. Cell-Cell Fusion

Cell-to-cell fusion assay was performed as previously described ^18;46;47 with some minor modifications. MAGI-CCR5 cells, a HeLa cell line expressing CD4, CXCR4 and CCR5, and HIV-LTR-β-gal under control of HIV-1 Tat were used as target cells and HL 2/3 cells, a HeLa cell line which expresses HIV-1_HXB2 Env on the surface and Tat, Gag, Rev and Nef proteins in the cytoplasm but does not produce detectable mature virions⁴⁸ were used as effector cells.

Following fusion of the two cell types, Tat produced by the HL2/3 cells activates β-gal expression in Magi cells. Briefly, MAGI-CCR5 cells were plated in a black 96-well plate with clear bottom at 1.5 × 10⁴ /well and cultured for 4 hours at 37 °C. Next, the cells were incubated for 1 hour with escalating concentrations of NBD-compounds. HL 2/3 cells were then added to the culture at 10⁴ cells / well and incubated for 24 hours at 37 °C. β-gal expression was quantified with the Beta-Glo assay system (Promega) following the manufacturer’s instructions. The percent inhibition and the IC₅₀ values were calculated using the GraphPad Prism software.

4.2.7. CD4-Dependent cell-to-cell HIV-1 transmission inhibition assay

The CD4-dependent cell-to-cell HIV-1 transmission inhibition assay was performed as previously described with some modifications¹⁸. Briefly, target cells, GHOST (3) X4/R5 which are adherent cells, were plated at 10⁴ / well in a 96-well plate and cultured overnight. As transmitting cells we used 4×10³/well chronically infected H9/HIV-1_IIIB for the CXCR4-tropic assay and 2×10⁴ /well MOLT-4/CCR5 cells chronically infected with HIV-1_ADA for the CCR5-tropic assay. The transmitting cells, which are suspension cells, were treated with 200 μg/mL mitomycin C for 1 hour at 37 °C, washed with complete GHOST-medium and incubated with the target cells and drugs for 4 hours. The target cells were then washed three times with PBS and incubated with fresh medium for 20 hours. Cells were washed with PBS and lysed with 1% Triton X-100. Intracellular p24 contents were determined by ELISA.

Abbreviations

HIV-1

Human Immunodeficiency Virus Type 1

Env

Envelope

AIDS

acquire immunodeficiency syndrome

VSV-G

Vesicular stomatitis virus-G

HBTU

(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexauorophosphate)

TBSCl

tert-Butyldimethylsilyl chloride

DIPEA

N,N-Diisopropylethylamine

Alloc

allyloxycarbonyl

M_RCN

mass of recovered nitrile

brsm

based on recovered starting material