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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2010 May 28.
Published in final edited form as:
PMCID: PMC2736638
NIHMSID: NIHMS112956

Anti-AIDS Agents 78 . Design, Synthesis, Metabolic Stability Assessment, and Antiviral Evaluation of Novel Betulinic Acid Derivatives as Potent Anti-Human Immunodeficiency Virus (HIV) Agents

Abstract

In a continuing study of potent anti-HIV agents, seventeen 28,30-disubstituted betulinic acid (BA, 1) derivatives, as well as seven novel 3,28-disubstituted BA analogs were designed, synthesized, and evaluated for in vitro antiviral activity. Among them, compound 21 showed an improved solubility and equal anti-HIV potency (EC50: 0.09 μM), when compared to HIV entry inhibitors 3b (IC9564) and 4 (A43-D). Using a cyclic secondary amine to form the C-28 amide bond increased the metabolic stability of the derivatives significantly in pooled human liver microsomes. The most potent compounds 47 and 48 displayed potent anti-HIV activity with EC50 values of 0.007 μM and 0.006 μM, respectively. These results are slightly better than that of bevirimat (2), which is currently in Phase IIb clinical trials. Compounds 47 and 48 should serve as attractive promising leads to develop next generation, metabolically stable, 3,28-disubstituted bifunctional HIV-1 inhibitors as clinical trials candidates.

Introduction

As the world enters the third decade of the AIDS epidemic, this pandemic has rapidly grown into the fourth leading cause of mortality globally.1 Introduction of highly active antiretroviral therapy (HAART), which employs a combination of nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and/or protease inhibitors (PIs), has significantly improved the treatment of HIV/AIDS.2-5 However, the virus is suppressed rather than eradicated by HAART.6-8 On HAART regimens, multiple drug therapies can lead to increased adverse effects and toxicities due to long-term use and drug-drug interactions.9,10 Moreover, it inevitably leads to the emergence of multi-drug-resistant viral strains.11 In fact, a significant proportion of newly infected individuals harbor HIV-1 isolates that are resistant to at least one ART.12,13 Therefore, novel potent antiretroviral agents are needed, with different targets than currently approved drugs and preferably with simplified treatment regimens (fewer pills and less-frequent administration).

Triterpenes, such as betulinic acid (BA, 1)14, represent a promising class of anti-HIV agents with novel mechanisms. Two types of BA derivatives have exhibited potent anti-HIV profiles. C-3 esterification of BA led to the discovery of bevirimat (DSB, PA-457, 2)15, which is a HIV-1 maturation inhibitor (MI) that blocks cleavage of p25 (CA-SP1) to functional p24 (CA), resulting in the production of noninfectious HIV-1 particles.16,17 Bevirimat (2) is currently in Phase IIb clinical trials launched by Panacos Pharmaceuticals, Inc.18,19 On the other hand, the C-28 side chain was proven to be a necessary pharmacophore for anti-HIV entry activity, as seen with the equipotent diastereomers 3a (RPR103611)20,21 and 3b (IC9564)22,23. Mechanism of action studies have revealed that C-28 modified BA derivatives function at a post-binding, envelope-dependent step involved in fusion of the virus to the cell membrane.24 Recent studies further suggested that 3b may also function by targeting the V3 loop of gp120, a domain involved in chemokine receptor binding.25 Although 3a showed potent antiviral activity in vitro, the clinical development of 3a by Rhone-Poulenc (now Sanofi-Aventis) was stopped due to poor “pharmacodynamic properties”.26 However, the high potency and novel mechanism of 3a suggest that further modification of this compound class as HIV entry inhibitors is warranted. Therefore, in the current study, we mainly focused on the modification of BA to maintain the anti-HIV activity while improving pharmacokinetic properties.

Design

Structurally, the triterpenoid skeleton of BA contains three functional groups: C-3 hydroxyl group, C-28 carboxylic acid group and C-19 isopropenyl moiety. Because the C-19 isopropenyl group has been less investigated, modification was first carried out on this moiety. Previous research suggested that saturation of the 20(29) double bond did not influence the antiviral activity of the BA derivatives significantly.15 Therefore, we focused on the modification at the C-30 allyl position in order to better explore the SAR. Some early data suggested that thioether-linked substitution on the C-30 position retained the anti-HIV-1 potency slightly in the resulting BA analogs.27 In the current study, the bioisosteric oxygen ether linker was chosen to replace the thioether group. Leucine and 8-aminooctanoic acid were proved to improve entry inhibition in our previous study, thus they were incorporated into the C-28 side chain of the diverse C-30 modified analogs to yield compounds 10-30.

The fact that 2 has suitable pharmocokinetic properties for development, but 3a does not, led us to speculate that the C-28 side chain, which is critical for anti-HIV entry activity, may be responsible for the poor performance of 3a in pre-clinical testing. Indeed, in our study, we observed that C-28 modified BA derivatives, including 4 (A43-D)28 which is the prior best anti-entry hit, are less water-soluble. In the meanwhile, although 2 has been demonstrated to be metabolized primarily by UGT glucuronidation,29 there is no report regarding to the metabolism of C-28 modified BA analogs. Therefore, in the present study, in vitro metabolic stability assessment was first carried out in pooled human liver microsomes (BD Biosciences), which contain enzymes including cytochrome P450 (CYPs), UGTs, and FMO, etc. The results revealed that C-28 modified BA derivatives like 4 are quickly metabolized in liver microsomes. Therefore, novel C-28 side chains were designed and synthesized. A cyclic secondary amine (from 4-piperidine butyric acid), rather than a primary amine, was used to form the critical amide bond with the C-28 carboxylic acid group, which yielded 38.

Moreover, although BA derivatives with both C-3 ester and C-28 amide side chains exhibit both anti-entry and maturation activity,30 they also display some metabolic problems, likely due to the C-28 side chain. Thus, modifications were also carried out on C-28 and C-3 side chains to enhance the stability as well as increase the anti-HIV activity in the 3,28-disubstituted BA analogs, which yielded compounds 36, 39-40 and 45-48. This article reports the design, syntheses, SAR, and metabolic stability assessment of these novel BA derivatives as potent HIV-1 inhibitors.

Chemistry

Scheme 1 depicts the synthetic pathway of 28,30-disubstituted BA derivatives 10-30. The C-3 β-hydroxyl group of BA was protected as the acetate ester of 3-O-Ac-BA (5). Compound 5 was reacted with oxalyl chloride in dichloromethane to yield an intermediate acid chloride, which was then treated with leucine methyl ester or 8-aminooctanoic acid methyl ester to furnish 6 and 7. Allylic bromination of 6 and 7 was carried out using N-bromosuccinimide (NBS) in dilute acetonitrile at room temperature to provide 30-bromo BA derivatives 8 and 9. The bromide group of 8 and 9 was then substituted by a diverse set of nucleophilic compounds to yield 10-20. In this step, the desired nucleophilic compound was first treated with 10 equivalents of NaH in THF for 30 min. The 30-bromo 8 or 9 was then reacted with the resulting suspension using a microwave apparatus at 120 °C. After cooling down, 1 mL MeOH-H2O was added to convert the intermediate esters to carboxylic acids by saponification, which furnished the target compounds 10-20 in 59-77% yields. Reaction of 20 with methylamine in the presence of HOBt/EDCI in dichloromethane led to 21 in a 69% yield. Saponification of the 30-bromo 8 and 9 with 2 N sodium hydroxide in MeOH/THF yielded the corresponding carboxylic acids 24-25. The previous reported 22-23 were also prepared by saponification of the ester intermediates 6 and 7. Reaction of silver acetate with 8 and 9, in the presence of a catalytic amount of the phase transfer catalyst tetrabutylammonium bromide (Bu4NBr) in acetonitrile, gave diacetoxy esters 26 and 27, which were then converted to 30-hydroxyl BA derivatives 28 and 29. The 3,28,30-trisubstituted analog 30 was acquired by reaction of 24 with 2,2-dimethylsuccinic anhydride in the presence of DMAP in pyridine.

Scheme 1
General synthetic route for 28,30-disubstituted BA derivatives

The 3,28-disubstituted 3β-amino analog 36 was successfully prepared as described in Scheme 2. Oxidation of 1 with 2 equivalents of PDC produced the 3-keto-BA 31 (87% yield). l-Leucine methyl ester was reacted with the C-28 carboxylic acid of 31 in the presence of DMAP and EDCI to furnish 32. The keto moiety of 32 was then converted to an oxime by treatment with hydroxylamine hydrochloride (NH2OH·HCl) in pyridine, which yielded 33 (90% yield).31 The 3β-amine 34 was readily prepared in 82% yield from oxime 33 by enantioselective reduction of the Schiff base with TiCl3 and NaCNBH3, as reported by Leeds and Kirst.32 Treatment of 34 with 2,2-dimethylsuccinic anhydride under DMAP in pyridine yielded 35. Finally, hydrolysis of 35 with 2 N sodium hydroxide furnished the desired 3,28-disubstituted 3β-amino BA analog 36.

Scheme 2
Synthesis of 3,28-disubstituted 3β-amino BA derivative

The syntheses of 3,28-disubstituted 28-piperidine analogs 38-48 were carried out according to Scheme 3. The 3-O-Ac-BA (5) was treated with oxalyl chloride in dichloromethane, followed by reaction with the readily prepared 4-piperidine butyric acid methyl ester to provide 37 in a 94% yield. Saponification of 37 yielded the desired lead compound 38 quantitatively. Esterification of 38 with 2,2-dimethylsuccinic anhydride and 2,2-dimethylglutaric anhydride under DMAP in pyridine led to 39 and 40 in yields of 55% and 36%, respectively. The syntheses of 41-44 were carried out by reacting 38 with different amines in the presence of HOBt and EDCI. Reaction of the 3β-hydroxyl group of 41-44 with 2,2-dimethylsuccinic anhydride provided the 3,28-disubstituted target compounds 45-48 in yields of 58-81%.

Scheme 3
General synthetic route for 3,28-disubstituted 28-piperidine BA analogs

Results and Discussion

The anti-HIV-1 replication activities of the newly synthesized BA derivatives 10-30, 36, 38-40 and 45-48 were assessed in HIV-1IIIB infected MT-2 lymphocytes in parallel with AZT and 2. Compounds 21 and 45-48 were further evaluated against HIV-1NL4-3 in MT-4 cell lines and compared with 3b and 4. Because these two antiviral screening systems used slightly different protocols, the results may vary for the same compounds. The bioassay data are summarized in Tables 11 and and2,2, respectively, and the SAR conclusions are summarized in Figure 2.

Figure 2
Summary of SAR of BA analogs
Table 1
Anti-HIV-1 replication activities in HIV-1IIIB infected MT-2 cell linesa
Table 2
Anti-HIV-1 replication activities in HIV-1NL4-3 infected MT-4 cell linesb

Within the 28,30-disubstituted BA derivatives, the C-28 leucine, C-30 substituted analogs 10-19, 24 and 28 did not inhibit viral replication. Because the monosubstituted C-28 leucine derivative 22 did not exhibit antiviral replication activity either, we postulated that the derivatives did not lose antiviral potency due to the C-30 modification. However, because the presence of different C-30 substitutions did not increase the anti-HIV-1 activity, the C-19 isopropenyl moiety is unlikely to be an activity pharmacophore. Nevertheless, considering that BA derivatives with anti-entry necessary C-28 lipophilic side chains are less water-soluble, C-30 allylic modification may still be useful to influence pharmacokinetic properties, such as hydrophilicity and solubility.

The introduction of a free hydroxyl group at C-30 in 29 reduced the antiviral activity relative to 23 by several fold to an EC50 value of 14.8 μM. This result suggested that a hydrogen bond donor is not tolerated near the C-19 isopropenyl group; a conclusion that is also supported by prior data that a primary amine substituent at C-30 is unfavorable.27 In comparison, analogs with 2-morpholinoethoxy (20) and bromide (25) moieties at C-30 retained the antiviral activity of 23. Specifically, the 28-aminooctanoic acid derivatives 23, 20 and 25 showed antiviral EC50 values of 3.3 μM, 1.8 μM and 2.3 μM against HIV-1IIIB, respectively. These results demonstrated that the C-30 position of BA can accommodate some diverse ethers without decreasing the anti-HIV potency. Moreover, the introduction of the morpholinoethoxy moiety in 20, which reduced the Log P value of 23 from 9.79 to 8.26 (calculated by ACD/LogP DB software), resulted in an increase in the derivative’s solubility, confirming that the C-30 position may serve as a good place to incorporate water-solubilizing moieties.

Analog 21 with methylamine linked to the terminal carboxylic acid of 20 exhibited potent anti-HIV-1 activity with an EC50 value of 0.09 μM and TI of 250 against both HIV-1IIIB and HIV-1NL4-3 variants. These data are similar to those with the prior best entry inhibitor 4 (EC50: 0.10 μM, TI > 100). This result indicates that the amide moiety near the end of the C-28 side chain is necessary for enhanced antiviral potency. Interestingly, 21 differs from 4 in the direction of the terminal amide linkage (-CONHCH3 in 21 and -NHCOCH3 in 4). Compound 21 showed a significantly reduced Log P value of 7.5 compared with the lead compound 23 and prior best hit 4.

Analog 30 with a 3′,3′-dimethylsuccinyl side chain linked to the C-3 β-hydroxyl group of 24 showed extremely potent antiviral activity with an EC50 value of 0.011 μM and TI of 3.0×103, which are similar to those of 2 (EC50: 0.011 μM, TI > 3.6×103). This result suggests that the presence of small substitutions on C-30 of BA does not harm the high anti-HIV-1 potency of 2. Thus, incorporation of polar groups into the C-30 position may also help to improve the hydrophilicity of 3,28-disubstituted BA derivatives.

Because an amide is generally more stable in vivo than an ester moiety, we also synthesized 3,28-disubstituted 3β-amino BA derived analog 36. Its C-3 side chain is similar to that of 2, except for a C-3 amide rather than ester bond. However, the antiviral activity of 36 against HIV-1IIIB decreased significantly to an EC50 value of 13.2 μM, suggesting that bioisosteric replacement of the C-3 ester bond with an amide moiety is not tolerated.

Results from the metabolism study revealed that changing the C-28 side chain from 8-aminooctanoic acid (23) to 4-piperidine butyric acid (38) could significantly increase the in vitro metabolic stability. Specifically, approximately 50% of 23 disappeared after around 35 minutes of incubation with pooled human liver microsomes. A similar result was found with buspirone (t1/2 = 31 min), an established fast-metabolized drug used as reference in the same experiment, suggesting that 23 was degraded quite easily in the assay system. Comparatively, it took about 125 minutes to lose 50% of the newly designed analog 38, indicating a much longer half life (Table 3). This result might be due to the increased steric hindrance at the C-28 pharmacophore of 38, so that the amide bond would be less available to metabolic enzymes. Thus, 38 represents a more stable lead for the development of C-28 modified BA derived HIV-1 entry inhibitors and 3,28-disubstituted bifunctional inhibitors.

Table 3
In vitro metabolic stability of compounds 23 and 38c

From the bioassay data, we discovered that compound 39 with the C-3 side chain of 2 incorporated into 38, showed very potent anti-HIV-1 activity with an EC50 value of 0.015 μM and TI of 1.4 ×103, proving that the presence of a bulky amide moiety near C-28 does not reduce the antiviral potency of 2. Compound 40, with a 4′,4′-dimethylglutaryl rather than 3′,3′-dimethylsuccinyl C-3 ester side chain, had a higher antiviral EC50 value (0.23 μM), indicating the importance of the positioning of the dimethyl substitution in the C-3 modification of BA. However, compound 38 itself showed decreased antiviral activity compared with 23, likely due to the slightly reduced length of the new C-28 side chain compared with the previous 8-aminooctanoic chain (23). Indeed, the BA derivative with 7-aminoheptanoic acid as the C-28 side chain showed 16-fold decreased anti-HIV activity compared with 23,20 confirming the importance of the length of the C-28 side chain.

Compounds 47 and 48, which contain 3′,3′-dimethylsuccinyl at C-3 and a morpholine ring at the end of C-28 side chain that is separated from the terminal amide bond by a short alkyl spacer (two or three methylenes), exhibited extremely potent anti-HIV-1 replication activity against HIV-1IIIB with EC50 values of 0.007 μM and 0.006 μM, respectively, which are almost twofold better than that of 2. These two compounds were also two-to three-fold more potent than entry inhibitors 3b and 4 in the anti-HIV screening against HIV-1NL4-3. Compound 46, with morpholine directly involved in the amide bond, and 2 had equivalent anti-HIV-1IIIB potency (EC50: 0.011 μM). Compound 45 with methylamine at the end of C-28 showed a slightly decreased antiviral potency (EC50: 0.067 μM) against HIV-1IIIB compared with 2. These results further confirm the importance of the length of the C-28 side chain to the enhanced antiviral potency in 3,28-disubstituted BA analogs. The better potency of 47 and 48 indicates that the activity of 2 can be increased with proper C-28 substitution. Moreover, because the C-28 side chains in all prior potent HIV inhibitors terminate in a free carboxylic acid or amide, the success of 47 and 48 also demonstrates that other polar groups at the end of this side chain can also increase antiviral potency.

In conclusion, diverse 28,30-disubstituted BA analogs were synthesized in our study. We discovered that a hydrogen bond donor is not tolerated near the C-19 isopropenyl moiety. Otherwise, C-30 substitution did not significantly influence the anti-HIV-1 activity of BA derivatives. Therefore, the C-30 position serves as a good place to incorporate water-solubilizing moieties to increase the hydrophilicity. The resulting analog 21 showed a good solubility as well as equal potency against HIV-1 compared with the previous best anti-entry hit 4. Using a cyclic secondary amine moiety (piperidine) rather than a primary amine to form the C-28 amide bond significantly increased the metabolic stability of the derivatives in pooled human liver microsome assessment. Subsequent introduction of a second amide bond at the carboxylic terminus of this metabolically stable C-28 side chain and introduction of the 3′,3′-dimethylsuccinyl side chain at the C-3 position resulted in the discovery of 47 and 48, which showed extremely potent antiviral activity, slightly better than that of 2. They should serve as attractive promising leads for the development of a next generation of BA derived 3,28-disubstituted HIV-1 inhibitors, as clinical trial candidates.

Experimental Section

Chemistry

The melting points were measured with a Fisher Johns melting apparatus without correction. 1H NMR spectra were measured on a 300 MHz Varian Gemini 2000 spectrometer using Me4Si (TMS) as internal standard. The solvent used was CDCl3 unless otherwise indicated. Mass spectra were measured on Shimadzu LCMS-2010 (ESI-MS). High resolution mass spectra (HRMS) were measured on Shimadzu LCMS-IT-TOF with ESI interface. Elemental analyses were performed by Atlantic Microlab, Inc., Norcross, GA. Target compounds were analyzed for C, H and gave values within ± 0.4% of the theoretical values. HPLC for purity determinations were conducted using Shimadzu LCMS-2010 with a Grace Alltima 2.1mm × 100mm HP C18 3μ column and a Shimadzu SPD-M20A detector at 200 nm wavelength. Two different solvent systems for HPLC purity analyses were as follows: 1) solvent B: acetonitrile, solvent A: water with or without 0.1% formic acid, 2) solvent B: methanol, solvent A: water with or without 0.1% formic acid. The flow rate was 0.3 mL/min. The isocratic HPLC mode was used and the specific solvent percentage conditions for each tested compound were listed in the supporting information. All target compounds possessed a purity of at least 95% as determined by elemental analysis or by HPLC-UV-MS. Optical rotations were measured with a Jasco Dip-2000 digital polarimeter at 20 °C at the sodium D line. Thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC) were performed on Merck precoated silica gel 60 F-254 plates. Flash+™ and CombiFlash systems (Teledyn-Isco) were used as medium pressure column chromatography. Silica gel (200-400 mesh) from Aldrich, Inc., was used for column chromatography. All other chemicals were obtained from Aldrich, Inc.

3-O-Acetyl-betulinic acid (5)

A mixture of 1 (2.1 g), pyridine (1.5 mL) and acetic anhydride (Ac2O, 20 mL) was stirred at room temperature overnight until it became homogenous. The reaction was then poured into ice-cold water (30 mL) and stirred for 20 min. The crude product was filtered off and purified on a silica-gel column to yield 1.98 g (87% yield) of pure 5; white amorphous powder. Mp 289-291 °C. MS (ESI-) m/z: 497.38 (M- - H) for C32H50O4. 1H NMR (300 MHz, CDCl3): δ 4.74, 4.61 (1H each, s, H-29), 4.47 (1H, dd, J = 9.9, 5.9 Hz, H-3), 3.01 (1H, m, H-19), 2.05 (3H, s, OCOCH3), 1.69 (3H, s, H-30), 0.97, 0.93, 0.86, 0.84, 0.83 (3H each, s, 5 × CH3).

Syntheses of BA-derivatives 6, 7 and 37

Oxalyl chloride solution (2 M in CH2Cl2, 10 mL) was added to 5 (1 eq) in CH2Cl2 (10 mL) and stirred for 2 h. After concentration under vacuum, the residual mixture was treated with leucine methyl ester (1.6 eq), 8-aminooctanoic acid methyl ester (1.6 eq), or 4-piperidine butyric acid methyl ester (1.6 eq) and triethylamine (Et3N, 1.2 eq) in CH2Cl2. The mixture was stirred at room temperature overnight until no starting material was observed by TLC. The solution was then diluted with CH2Cl2 (20 mL) and washed three times with brine and distilled water. The organic layer was dried over anhydrous Na2SO4 and concentrated to dryness under reduced pressure. The residue was chromatographed using a silica gel column to yield the pure target compounds.

Methyl N-[3β-acetoxy-lup-20(29)-en-28-oyl]-leucinate (6)

1.15 g (80.5% yield) starting from 1 g of 5; white amorphous powder. Mp 230-232 °C. MS (ESI+) m/z: 626.48 (M+ + H), 648.47 (M+ + Na) for C39H63NO5. 1H NMR (300 MHz, CDCl3): δ 5.87 (1H, d, J = 8 Hz, -CONH-), 4.72, 4.59 (1H each, s, H-29), 4.64 (1H, m, -NHCH-), 4.49 (1H, t, J = 8 Hz, H-3), 3.73 (3H, s, -COOCH3), 3.05 (1H, m, H-19), 2.10-2.20 (1H, m, H-13), 2.04 (3H, s, OCOCH3), 1.68 (3H, s, H-30), 1.01 (6H, s, leucine moiety -(CH3)2), 0.97 (6H, s, 2 × CH3), 0.89, 0.84, 0.83 (3H each, s, 3 × CH3).

Methyl N-[3β-acetoxy-lup-20(29)-en-28-oyl]-8-aminooctanoate (7)

643 mg (98% yield) starting from 500 mg of 5; light yellow amorphous powder. Mp 104-105 °C. MS (ESI+) m/z: 654.5 (M+ + H) for C41H67NO5. 1H NMR (300 MHz, CDCl3): δ 5.57 (1H, t, J = 6 Hz, -CONH-), 4.73, 4.60 (1H each, s, H-29), 4.45 (1H, m, H-3), 3.67 (3H, s, -COOCH3), 3.30-3.08 (3H, m, H-19, -CONHCH2-), 2.50 (1H, m, H-13), 2.31 (2H, t, J =7 Hz, -CH2COOCH3), 2.05 (3H, s, OCOCH3), 1.68 (3H, s, H-30), 0.97, 0.94 (3H each, s, 2 × CH3), 0.85, 0.84, 0.81 (3H each, s, 3 × CH3).

Methyl N-[3β-acetoxy-lup-20(29)-en-28-oyl]-4-piperidine butanoate (37)

1.02 g (94% yield) starting from 800 mg of 5; white amorphous powder. Mp 195-197 °C. MS (ESI+) m/z: 666.5 (M+ + H) for C42H67NO5. 1H NMR (300 MHz, CDCl3): δ 4.72, 4.57 (1H each, s, H-29), 4.47 (1H, dd, J = 11.1, 5.7 Hz, H-3), 3.67 (3H, s, -COOCH3), 3.67-3.47 (4H, m, 28-CON(CH2CH2)2CH-), 2.99 (1H, m, H-19), 2.31 (2H, t, J =7 Hz, -CH2COOCH3), 2.05 (3H, s, OCOCH3), 1.68 (3H, s, H-30), 0.96 (6H, s, 2 × CH3), 0.94, 0.82, 0.75 (3H each, s, 3 × CH3).

Syntheses of BA-derivatives 8 and 9

A mixture of N-bromosuccinimide (1.1 eq) and 6 or 7 (1 eq) in acetonitrile (ACN, 30 mL) was stirred at room temperature until the starting material was not observed by TLC. The reaction was concentrated to dryness under reduced pressure and chromatographed over silica gel to yield pure target compounds.

Methyl N-[3β-acetoxy-30-bromo-lup-20(29)-en-28-oyl]-leucinate (8)

297 mg (66% yield) starting from 400 mg of 6; light yellow amorphous powder. Mp 127-129 °C. MS (ESI+) m/z: 704.4 (M+ + H), 706.4 (M+ + H) for C39H62BrNO5. 1H NMR (300 MHz, CDCl3): δ 5.81 (1H, d, J = 8 Hz, -CONH-), 5.11, 5.05 (1H each, s, H-29), 4.65 (1H, m, -NHCH-), 4.45 (1H, t, J = 8 Hz, H-3), 4.00 (2H, s, H2-30), 3.72 (3H, s, -COOCH3), 3.10 (1H, m, H-19), 2.50-2.32 (1H, m, H-13), 2.05 (3H, s, OCOCH3), 1.02 (6H, s, leucine moiety -(CH3)2), 0.99 (6H, s, 2 × CH3), 0.89, 0.86, 0.85 (3H each, s, 3 × CH3).

Methyl N-[3β-acetoxy-30-bromo-lup-20(29)-en-28-oyl]-8-aminooctanoate (9)

402 mg (72.5% yield) starting from 360 mg of 7; light yellow amorphous powder. Mp 99-101 °C. MS (ESI+) m/z: 732.4 (M+ + H), 734.4 (M+ + H) for C41H66BrNO5. 1H NMR (300 MHz, CDCl3): δ 5.59 (1H, t, J = 6 Hz, -CONH-), 5.13, 5.04 (1H each, s, H-29), 4.47 (1H, t, J = 8.1 Hz, H-3), 4.00 (2H, s, H2-30), 3.67 (3H, s, -COOCH3), 3.41-3.09 (3H, m, H-19, -CONHCH2-), 2.46 (1H, m, H-13), 2.31 (2H, t, J=7.5 Hz, -CH2COOCH3), 2.04 (3H, s, OCOCH3), 0.97, 0.93 (3H each, s, 2 × CH3), 0.89, 0.84, 0.83 (3H each, s, 3 × CH3).

Syntheses of BA-derivatives 10-20

NaH (60% in mineral oil) was washed three times with hexane. A solution of appropriate nucleophilic compound (8 eq) and NaH (10 eq) in anhydrous THF (1.5 mL) was stirred under dry nitrogen at room temperature for 30 min. The 30-bromo BA derivative 8 or 9 (1 eq) was then added into the system. The reaction was heated using microwave (Biotage) at 120 °C for 30 min. After cooling to room temperature, 1 mL MeOH-H2O was added into the mixtures and stirred to transform the intermediate esters to carboxylic acids by saponification. The reaction was neutralized with 10% HCl and dried under vacuum and reconstituted with EtOAc. The organic layer was washed with brine and dried over anhydrous Na2SO4 and concentrated to dryness under reduced pressure. The residue was chromatographed using a silica gel column to yield the pure target compounds.

N-[3β-Hydroxy-30-ethoxy-lup-20(29)-en-28-oyl]-leucine (10)

22 mg (59% yield) starting from 40 mg of 8; white amorphous powder. Mp 128-130 °C. MS (ESI-) m/z: 612.4 (M- - H) for C38H63NO5. 1H NMR (300 MHz, CDCl3): δ 5.88 (1H, d, J = 8 Hz, -CONH-), 4.93, 4.92 (2H, br s, H-29), 4.63-4.58 (1H, m, -NHCH-), 3.90 (2H, s, H2-30), 3.47 (2H, m, 30-OCH2CH3), 3.18 (1H, dd, J = 11.1, 5.4 Hz, H-3), 2.99 (1H, m, H-19), 2.50-2.32 (1H, m, H-13), 1.00 (9H, br s, 30-OCH2CH3, leucine moiety -(CH3)2), 0.96 (6H, s, 2 × CH3), 0.89, 0.86, 0.85 (3H each, s, 3 × CH3).

N-[3β-Hydroxy-30-propoxy-lup-20(29)-en-28-oyl]-leucine (11)

23 mg (60% yield) starting from 40 mg of 8; white amorphous powder. Mp 116-117 °C. MS (ESI-) m/z: 626.5 (M- - H) for C39H65NO5. 1H NMR (300 MHz, CDCl3): δ 6.13 (1H, br s, -CONH-), 4.91, 4.90 (2H, br s, H-29), 4.52 (1H, m, -NHCH-), 3.90 (2H, s, H2-30), 3.36 (2H, t, J = 6.9 Hz, 30-OCH2CH2CH3), 3.18 (1H, dd, J = 11.1, 5.4 Hz, H-3), 2.99 (1H, m, H-19), 0.96, 0.94, 0.92, 0.89 (15H, m, 30-O(CH2)2CH3, leucine moiety -(CH3)2, CH3-23, 24), 0.82, 0.81, 0.79 (3H each, s, 3 × CH3).

N-[3β-Hydroxy-30-butoxy-lup-20(29)-en-28-oyl]-leucine (12)

10 mg (37% yield) starting from 30 mg of 8; yellow amorphous powder. Mp 104-105 °C. MS (ESI-) m/z: 640.2 (M- - H) for C40H67NO5. 1H NMR (300 MHz, CDCl3): δ 6.01 (1H, br s, -CONH-), 4.90, 4.88 (2H, br s, H-29), 4.58 (1H, m, -NHCH-), 3.89 (2H, s, H2-30), 3.37 (2H, m, 30-OCH2(CH2)2CH3), 3.17 (1H, m, H-3), 3.01 (1H, m, H-19), 0.99 (9H, br s, 30-O(CH2)3CH3, leucine moiety -(CH3)2), 0.96 (6H, s, 2 × CH3), 0.86, 0.84, 0.81 (3H each, s, 3 × CH3).

N-[3β-Hydroxy-30-phenethoxy-lup-20(29)-en-28-oyl]-leucine (13)

37 mg (77% yield) starting from 50 mg of 8; light yellow amorphous powder. Mp 155-157 °C. MS (ESI-) m/z: 688.4 (M- - H). 1H NMR (300 MHz, CDCl3): δ 7.68-7.62 (2H, m, H ar-3′), 7.28-7.20 (3H, m, H ar-2′, 4′), 5.97 (1H, br s, -CONH-), 4.91, 4.90 (2H, br s, H-29), 4.44 (1H, m, -NHCH-), 3.93 (2H, s, H2-30), 3.64 (2H, t, J = 7.2 Hz, 30-OCH2CH2Ph), 3.17 (1H, dd, J = 11.1, 5.4 Hz, H-3), 2.91 (1H, m, H-19), 2.57 (2H, m, 30-OCH2CH2Ph), 0.95 (12H, s, leucine moiety -(CH3)2, CH3-23, 24), 0.89, 0.78, 0.74 (3H each, s, 3 × CH3). Anal. (C44H67O5N · 2H2O) C, H, O.

N-[3β-Hydroxy-30-(4′-methoxyphenethoxy)-lup-20(29)-en-28-oyl]-leucine (14)

46 mg (64% yield) starting from 70 mg of 8; light yellow amorphous powder. Mp 128-129 °C. MS (ESI-) m/z: 718.5 (M- - H). 1H NMR (300 MHz, CDCl3): δ 7.27, 7.16-7.13, 6.85-6.82 (5H, m, H ar-2′, 3′, 4′), 5.97 (1H, br s, -CONH-), 4.91, 4.89 (H each, br s, H-29), 4.48 (1H, m, -NHCH-), 3.93 (2H, s, H2-30), 3.79 (3H, s, ar-OCH3), 3.60 (2H, t, J = 7.2 Hz, 30-OCH2CH2Ph(p-OCH3)), 3.17 (1H, dd, J = 11.1, 5.4 Hz, H-3), 2.85 (1H, t, J = 7.5 Hz, H-19), 2.39 (3H, m, 30-OCH2CH2Ph(p-OCH3), H-13), 0.95, 0.93, 0.90 (15H, s, leucine moiety -(CH3)2, 3 × CH3), 0.79, 0.75 (3H each, s, 2 × CH3). Anal. (C45H69 O6N · 4½H2O) C, H, O.

N-[3β-Hydroxy-30-(4′-fluorophenethoxy)-lup-20(29)-en-28-oyl]-leucine (15)

24 mg (40% yield) starting from 60 mg of 8; light yellow amorphous powder. Mp 102-104 °C. MS (ESI-) m/z: 706.4 (M- - H). 1H NMR (300 MHz, CDCl3): δ 7.16-6.80 (5H, m, H ar-2′, 3′, 4′), 5.96 (1H, br s, -CONH-), 4.90, 4.89 (2H, br s, H-29), 4.48 (1H, m, -NHCH-), 3.92 (2H, s, H2-30), 3.61 (2H, t, J = 7.2 Hz, 30-OCH2CH2Ph(p-F)), 3.17 (1H, m, H-3), 2.87 (1H, t, J = 7.5 Hz, H-19), 2.36-2.10 (3H, m, 30-OCH2CH2Ph(p-F), H-13), 0.95, 0.88 (15H, s, leucine moiety -(CH3)2, 3 × CH3), 0.75, 0.73 (3H each, s, 2 × CH3). Anal. (C44H66 O5NF · 3½H2O) C, H, O.

N-[3β-Hydroxy-30-(4′-bromophenethoxy)-lup-20(29)-en-28-oyl]-leucine (16)

18 mg (41% yield) starting from 40 mg of 8; light yellow amorphous powder. Mp 127-129 °C. MS (ESI-) m/z: 706.4 (M- - H). 1H NMR (300 MHz, CDCl3): δ 7.56-7.28 (5H, m, H ar-2′, 3′, 4′), 5.96 (1H, br s, -CONH-), 4.91, 4.90 (2H, br s, H-29), 4.48 (1H, m, -NHCH-), 3.91 (2H, s, H2-30), 3.60 (2H, t, J = 7.0 Hz, 30-OCH2CH2Ph(p-Br)), 3.17 (1H, dd, J = 11.0, 5.6 Hz, H-3), 2.89 (1H, t, J = 7.5 Hz, H-19), 2.39 (1H, m, 30-OCH2CH2Ph(p-Br)), 0.96 (12H, s, leucine moiety -(CH3)2, 2 × CH3), 0.82, 0.79, 0.75 (3H each, s, 3 × CH3). Anal. (C44H66 O5NBr · 2H2O) C, H, O.

N-[3β-Hydroxy-30-(4′-chlorophenethoxy)-lup-20(29)-en-28-oyl]-leucine (17)

16 mg (38% yield) starting from 40 mg of 8; light yellow amorphous powder. Mp 119-121 °C. MS (ESI-) m/z: 706.4 (M- - H). 1H NMR (300 MHz, CDCl3): δ 7.18-6.87 (5H, m, H ar-2′, 3′, 4′), 5.96 (1H, br s, -CONH-), 4.91, 4.90 (2H, br s, H-29), 4.48 (1H, m, -NHCH-), 3.91 (2H, s, H2-30), 3.62 (2H, t, J = 6.8 Hz, 30-OCH2CH2Ph(p-Cl)), 3.17 (1H, dd, J = 11.0, 5.6 Hz, H-3), 2.87 (1H, t, J = 7.5 Hz, H-19), 2.36-2.06 (3H, m, 30-OCH2CH2Ph(p-Cl), H-13), 0.96 (15H, s, leucine moiety -(CH3)2, 3 × CH3), 0.81, 0.76 (3H each, s, 2 × CH3). Anal. (C44H66 O5NCl · H2O) C, H, O.

N-[3β-Hydroxy-30-morpholino-lup-20(29)-en-28-oyl]-leucine (18)

22 mg (41% yield) starting from 60 mg of 8; white amorphous powder. Mp 98-100 °C. MS (ESI-) m/z: 653.5 (M- - H). 1H NMR (300 MHz, CDCl3): δ 5.61 (1H, d, J = 6 Hz, -CONH-), 4.92, 4.90 (H each, s, H-29), 4.63-4.58 (1H, m, -NHCH-), 3.72 (4H, m, 30-N(CH2CH2)2O), 3.17 (1H, dd, J = 11.1, 5.4 Hz, H-3), 3.00 (3H, m, H-19, H2-30), 2.53 (4H, m, 30-N(CH2CH2)2O), 2.42 (1H, m, H-13), 0.96 (6H, s, leucine moiety -(CH3)2), 0.92 (6H, s, 2 × CH3), 0.86, 0.81, 0.75 (3H each, s, 3 × CH3). Anal. (C40H66 O5N2 · 2H2O) C, H, O.

N-[3β-Hydroxy-30-(2′-morpholinoethoxy)-lup-20(29)-en-28-oyl]-leucine (19)

26 mg (56% yield) starting from 50 mg of 8; white amorphous powder. Mp 89-91 °C. MS (ESI-) m/z: 697.4 (M- - H). 1H NMR (300 MHz, CDCl3): δ 5.61 (1H, d, J = 8 Hz, -CONH-), 4.92, 4.90 (H each, s, H-29), 4.59 (1H, m, -NHCH-), 3.94 (2H, s, H2-30), 3.72 (4H, m, -N(CH2CH2)2O), 3.58 (2H, t, J = 5.7 Hz, 30-OCH2CH2-morpholine), 3.18 (1H, dd, J = 11.4, 4.6 Hz, H-3), 3.01 (1H, m, H-19), 2.60 (2H, t, J = 5.4 Hz, 30-OCH2CH2-morpholine), 2.53 (4H, m, -N(CH2CH2)2O), 1.00 (6H, s, leucine moiety - (CH3)2), 0.96 (6H, s, 2 × CH3), 0.89, 0.85, 0.80 (3H each, s, 3 × CH3). Anal. (C42H70 O6N2 · H2O) C, H, O.

N-[3β-Hydroxy-30-(2′-morpholinoethoxy)-lup-20(29)-en-28-oyl]-8-aminooctanoic acid (20)

51 mg (64% yield) starting from 80 mg of 9; white amorphous powder. Mp 111-112 °C. MS (ESI-) m/z: 725.5 (M- - H) for C44H74N2O6. 1H NMR (300 MHz, CDCl3): δ 5.61 (1H, d, J = 8 Hz, -CONH-), 4.91, 4.90 (H each, s, H-29), 3.94 (2H, s, H2-30), 3.72 (4H, m, -N(CH2CH2)2O), 3.58 (2H, t, J = 5.7 Hz, 30-OCH2CH2-morpholine), 3.18 (3H, m, -CONHCH2-, H-3), 3.01 (1H, m, H-19), 2.60 (2H, t, J = 5.4 Hz, 30-OCH2CH2-morpholine), 2.53 (4H, m, -N(CH2CH2)2O), 2.28 (2H, t, J =7.5 Hz, -CH2COOH), 0.96 (6H, s, 2 × CH3), 0.92, 0.81, 0.75 (3H each, s, 3 × CH3).

Syntheses of BA-derivatives 26 and 27

A solution of 30-bromo BA derivative 8 or 9 (1 eq), silver acetate (AgOAc, 2 eq) and tetrabutylammonium bromide (Bu4NBr, 0.2 eq) in acetonitrile (1.5 mL) was heated using microwave at 100 °C for 25 min. The precipitant was filtered and the solution was concentrated to dryness under vacuum. The residue was chromatographed over a silica-gel column to yield the pure diacetoxy intermediates 26 and 27.

Methyl N-[3β,30-diacetoxy-lup-20(29)-en-28-oyl]-leucinate (26)

80 mg (68% yield) starting from 120 mg of 117; off-white amorphous powder. Mp 201-203 °C. MS (ESI+) m/z: 684.5 (M+ + H) for C41H65NO7. 1H NMR (300 MHz, CDCl3): δ 5.69 (1H, br s, -CONH-), 4.97, 4.94 (2H, d, J = 9, H-29), 4.58-4.52 (3H, m, -NHCH-, H2-30), 4.45 (1H, t, J = 8 Hz, H-3), 3.72 (3H, s, -COOCH3), 3.10 (1H, m, H-19), 2.50-2.32 (1H, m, H-13), 2.08 (6H, s, 2 × OCOCH3), 1.05 (6H, s, leucine moiety -(CH3)2), 0.96 (6H, s, 2 × CH3), 0.89, 0.82, 0.81(3H each, s, 3 × CH3).

Methyl N-[3β,30-diacetoxy-lup-20(29)-en-28-oyl]-8-aminooctanoate (27)

77.8 mg (69.5% yield) starting from 80 mg of 118; white amorphous powder. Mp 167-169 °C. MS (ESI+) m/z: 712.5 (M+ + H) for C43H69NO7. 1H NMR (300 MHz, CDCl3): δ 5.60 (1H, t, J = 4.6 Hz, -CONH-), 4.94, 4.90 (1H each, s, H-29), 4.56 (2H, s, H2-30), 4.45 (1H, t, J = 7 Hz, H-3), 3.66 (3H, s, -COOCH3), 3.41-3.09 (3H, m, H-19, -CONHCH2-), 2.46 (1H, m, H-13), 2.31 (2H, t, J=7.5 Hz, -CH2COOCH3), 2.05 (6H, s, 2 × OCOCH3), 0.97, 0.96, 0.85, 0.81, 0.80 (3H each, s, 5 × CH3).

Syntheses of BA-derivatives 22-25, 28-29, 36 and 38

To a solution of the appropriate ester intermediates 6-9, 26-27, 35 and 37 (1 eq) in MeOH (8 mL) and THF (4 mL) was added 2 N NaOH (4 mL). The mixture was stirred overnight, and then neutralized with 20% HCl. The solution was dried under vacuum and reconstituted with EtOAc. The organic layer was washed with brine and dried over anhydrous Na2SO4 and concentrated to dryness under reduced pressure. The residue was chromatographed using a silica gel column to yield the pure target compounds.

N-[3β-Hydroxy-lup-20(29)-en-28-oyl]-leucine (22)

27 mg (100% yield) starting from 30 mg of 6; white amorphous powder. Mp 243-244 °C. MS (ESI-) m/z: 568.42 (M- - H) for C36H59NO4. 1H NMR (300 MHz, CDCl3): δ 5.86 (1H, d, J = 8 Hz, -CONH-), 5.11, 5.02 (1H each, s, H-29), 4.65 (1H, m, -NHCH-), 3.17 (1H, dd, J = 9.7, 5.4 Hz, H-3), 3.10-3.03 (1H, m, H-19), 1.68 (3H, s, H-30), 1.00 (6H, s, leucine moiety -(CH3)2), 0.96 (6H, s, 2 × CH3), 0.83, 0.80, 0.79 (3H each, s, 3 × CH3). [α]25D -17.2 ° (c = 1.40, CHCl3).

N-[3β-Hydroxy-lup-20(29)-en-28-oyl]-8-aminooctanoic acid (23)

37 mg (100% yield) starting from 40 mg of 7; white amorphous powder. Mp 110-112 °C. MS (ESI-) m/z: 596.5 (M- - H) for C38H63NO4. 1H NMR (300 MHz, CDCl3): δ5.60 (1H, br s, -CONH-), 4.73, 4.60 (1H each, s, H-29), 3.21-3.09 (4H, m, H-3, H-19, -CONHCH2-), 2.31 (2H, t, J = 6.9 Hz, -CH2COOH), 2.10-2.20 (1H, m, H-13), 1.68 (3H, s, H-30), 0.97 (6H, s, 2 × CH3), 0.85, 0.79, 0.75 (3H each, s, 3 × CH3). [α]25D -3.6 ° (c = 0.19, CHCl3). [α]25D -8.48 ° (c = 0.20, MeOH).

N-[3β-Hydroxy-30-bromo-lup-20(29)-en-28-oyl]-leucine (24)

102 mg (100% yield) starting from 110 mg of 8; white amorphous powder. Mp 102-104 °C. MS (ESI-) m/z: 646.41, 648.39 (M- - H) for C36H58BrNO4. 1H NMR (300 MHz, CDCl3): δ 5.86 (1H, d, J = 8 Hz, -CONH-), 5.11, 5.02 (1H each, s, H-29), 4.65 (1H, m, -NHCH-), 3.90 (2H, s, H2-30), 3.17 (1H, dd, J = 9.7, 5.4 Hz, H-3), 3.10-3.03 (1H, m, H-19), 1.00 (6H, s, leucine moiety -(CH3)2), 0.96 (6H, s, 2 × CH3), 0.83, 0.80, 0.79 (3H each, s, 3 × CH3). [α]25D -10.5 ° (c = 0.15, MeOH).

N-[3β-Hydroxy-30-bromo-lup-20(29)-en-28-oyl]-8-aminooctanoic acid (25)

44 mg (95% yield) starting from 50 mg of 9; white amorphous powder. Mp 119-122 °C. MS (ESI-) m/z: 674.4 (M- - H) for C38H62BrNO4. 1H NMR (300 MHz, CDCl3): δ5.60 (1H, br s, -CONH-), 5.13, 5.04 (1H each, s, H-29), 4.00 (2H, s, H2-30), 3.21-3.09 (4H, m, H-3, H-19, -CONHCH2-), 2.34 (2H, m, -CH2COOH), 0.96 (6H, s, 2 × CH3), 0.92, 0.82, 0.81 (3H each, s, 3 × CH3). [α]25D -16.5 ° (c = 0.22, MeOH).

N-[3β,30-Dihydroxy-lup-20(29)-en-28-oyl]-leucine (28)

24 mg (98% yield) starting from 30 mg of 26; white amorphous powder. Mp 145-148 °C. MS (ESI-) m/z: 584.5 (M- - H) for C36H59NO5. 1H NMR (300 MHz, CDCl3): δ 5.89 (1H, br s, -CONH-), 4.91, 4.90 (1H each, s, H-29), 4.68 (1H, m, -NHCH-), 4.12 (2H, s, H2-30), 3.17 (1H, dd, J = 11.2, 5.6 Hz, H-3), 3.01 (1H, m, H-19), 2.34 (1H, m, H-13), 1.10 (6H, s, leucine moiety -(CH3)2), 0.99 (6H, s, 2 × CH3), 0.86, 0.83, 0.80 (3H each, s, 3 × CH3). [α]25D -39.3 ° (c = 0.35, MeOH).

N-[3β,30-Dihydroxy-lup-20(29)-en-28-oyl]-8-aminooctanoic acid (29)

50 mg (80% yield) starting from 58 mg of 27; white amorphous powder. Mp 135-137 °C. MS (ESI-) m/z: 612.5 (M- - H) for C38H63NO5. 1H NMR (300 MHz, CDCl3): δ5.61 (1H, br s, -CONH-), 4.94, 4.90 (1H each, s, H-29), 4.12 (2H, s, H2-30), 3.25-3.15 (3H, m, H-3, -CONHCH2-), 3.01 (1H, m, H-19), 2.34 (2H, t, J = 7.6 Hz, -CH2COOH), 2.06 (1H, m, H-13), 0.97, 0.96 (3H each, s, 2 × CH3), 0.92, 0.82, 0.75 (3H each, s, 3 × CH3). [α]25D -143.3 ° (c = 0.10, MeOH).

N’-[3β-N-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-leucine (36)

24 mg (98%) starting from 25 mg of 35; white amorphous powder. Mp 248-250 °C. MS (ESI-) m/z: 695.5 (M- - H) for C42H68N2O6. 1H NMR (300 MHz, CDCl3): δ 5.87 (1H, d, J = 7.6 Hz, -CONH-), 4.72, 4.58 (1H each, s, H-29), 4.64 (1H, m, -NHCH-), 3.59 (1H, m, H-3), 2.99 (1H, m, H-19), 2.64-2.42 (2H, m, H-2′), 1.68 (3H, s, H-30), 1.30, 1.26 (3H each, s, 2 × CH3-3′), 1.00 (6H, s, leucine moiety -(CH3)2), 0.96 (6H, s, 2 × CH3), 0.89, 0.86, 0.85 (3H each, s, 3 × CH3). [α]25D -16.1 ° (c = 0.28, MeOH).

N-[3β-Hydroxy-lup-20(29)-en-28-oyl]-4-piperidine butyric acid (38)

190 mg (100%) starting from 200 mg of 37, white amorphous powder. Mp 145-146 °C. MS (ESI-) m/z: 608.4 (M- - H) for C39H63NO4. 1H NMR (300 MHz, CDCl3): δ 4.72, 4.57 (1H each, s, H-29), 3.67-3.47 (4H, m, 28-CON(CH2CH2)2CH-), 3.19 (1H, m, H-3), 2.99 (1H, m, H-19), 2.31 (2H, t, J = 8.4 Hz, -CH2COOH), 1.68 (3H, s, H-30), 0.96 (6H, s, 2 × CH3), 0.94, 0.82, 0.81 (3H each, s, 3 × CH3). [α]25D -22.7 ° (c = 0.33, MeOH).

Synthesis of BA-derivatives 21 and 41-44

A solution of 20 or 38 (1 eq), EDCI (2 eq), N-Hydroxybenzotriazole (HOBt, 1 eq), Et3N (0.05 mL) and the appropriate amine (2 eq) in anhydrous CH2Cl2 (8 mL) was stirred at room temperature overnight until the starting material was not observed by TLC. The solution was diluted with CH2Cl2 (20 mL) and washed three times with brine and distilled water. The organic layer was dried over anhydrous Na2SO4 and concentrated to dryness under reduced pressure. The residue was chromatographed using a silica gel column to yield pure target compounds.

β-Hydroxy-30-(2′-morpholinoethoxy)-lup-20(29)-en-28-oyl]-8-aminooctanoyl]-aminomethane (21)

21 mg (69% yield) starting from 30 mg of 20; white amorphous powder. Mp 106-107 °C. MS (ESI+) m/z: 740.5 (M+ + H) for C45H77 N3O5. 1H NMR (300 MHz, CDCl3): δ 5.61 (2H, m, 2 × -CONH-), 4.92, 4.90 (H each, s, H-29), 3.94 (2H, s, H2-30), 3.72 (4H, m, -N(CH2CH2)2O), 3.58 (2H, t, J = 5.7 Hz, 30-OCH2CH2-morpholine), 3.28-3.14 (3H, m, -CONHCH2-, H-3), 3.01 (1H, m, H-19), 2.81 (3H, d, J = 4.8 Hz, -CONHCH3), 2.60 (2H, t, J = 5.4 Hz, 30-OCH2CH2-morpholine), 2.53 (4H, m, -N(CH2CH2)2O), 2.16 (2H, t, J =7.5 Hz, -CH2CONHCH3), 0.96 (6H, s, 2 × CH3), 0.92, 0.81, 0.75 (3H each, s, 3 × CH3).

N’-[N-[3β-Hydroxy-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]-aminomethane (41)

46 mg (100% yield) starting from 50 mg of 38, off-white amorphous powder. Mp 202-204 °C. MS (ESI+) m/z: 623.5 (M+ + H) for C40H66N2O31H NMR (300 MHz, CDCl3): δ 5.44 (1H, br s, -CONHCH3), 4.69, 4.54 (1H each, s, H-29), 3.58-3.54 (4H, m, 28-CON(CH2CH2)2CH-), 3.16 (1H, m, H-3), 2.95 (1H, m, H-19), 2.78 (3H, d, J = 4.8 Hz, -CONHCH3), 2.16 (2H, t, J = 7.5 Hz, -CH2CONHCH3), 1.66 (3H, s, H-30), 0.93 (6H, s, 2 × CH3),0.92, 0.90, 0.79 (3H each, s, 3 × CH3). [α]25D -10.7 ° (c = 0.19, MeOH).

β-Hydroxy-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]-morpholine (42)

53 mg (87% yield) starting from 50 mg of 38, white amorphous powder. Mp 132-133 °C. MS (ESI+) m/z: 679.5 (M+ + H) for C43H70N2O4. 1H NMR (300 MHz, CDCl3): δ 4.69, 4.54 (1H each, s, H-29), 3.66-3.60 (8H, m, 28-CON(CH2CH2)2CH-, -CON(CH2CH2)2O), 3.42 (4H, m, -CON(CH2CH2)2O), 3.14 (1H, m, H-3), 2.95-2.60 (1H, m, H-19), 2.27 (2H, t, J = 9.2 Hz, -CH2CON(CH2CH2)2O), 1.65 (3H, s, H-30), 0.93 (3H each, s, 2 × CH3), 0.91, 0.83, 0.79 (3H each, s, 3 × CH3). [α]25D -20.0 ° (c = 0.31, MeOH).

N’-[N-[3β-Hydroxy-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]-2-aminoethylmorpholine (43)

54 mg (91% yield) starting from 55 mg of 38, white amorphous powder. Mp 114-116 °C. MS (ESI+) m/z: 722.6 (M+ + H), 744.5 (M+ + Na) for C45H75N3O4. 1H NMR (300 MHz, CDCl3): δ 6.79 (1H, br, s, -CONHCH2-), 4.68, 4.53 (1H each, s, H-29), 3.69-3.67 (8H, m, 28-CON(CH2CH2)2CH-, -CH2N(CH2CH2)2O), 3.31 (2H, m, -CONHCH2-), 3.17 (1H, m, H-3), 2.95-2.60 (1H, m, H-19), 2.43 (6H, m, -CH2N(CH2CH2)2O), 2.17 (2H, t, J = 7.5 Hz, -CH2CONHCH2-), 1.63 (3H, s, H-30), 0.93 (3H each, s, 2 × CH3), 0.90, 0.79, 0.72 (3H each, s, 3 × CH3). [α]25D -10.6 ° (c = 0.15, MeOH).

N’-[N-[3β-Hydroxy-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]-3-aminopropylmorpholine (44)

54 mg (90% yield) starting from 50 mg of 38, white amorphous powder. Mp 122-124 °C. MS (ESI+) m/z: 736.6 (M+ + H) for C46H77N3O4. 1H NMR (300 MHz, CDCl3): δ 5.93 (1H, br, s, -CONHCH2-), 3.15 (1H, m, H-3), 2.95-2.60 (1H, m, H-19), 2.47-2.41 (6H, m, -CH2N(CH2CH2)2O), 2.16 (2H, t, J = 7.5 Hz, -CH2CONHCH2-), 1.65 (3H, s, H-30), 0.93 (3H each, s, 2 × CH3), 0.91, 0.79, 0.72 (3H each, s, 3 × CH3). [α]25D -7.5 ° (c = 0.18, MeOH).

3-Deoxy-betulinic acid (31)

To a solution of 1 (2 g, 1 eq) in DMF was added pyridium dichromate (PDC, 2 eq). The mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with EtOAc (30 mL) and the precipitate was filtered through a short pack of Florisil. The solution was washed with 20% HCl and distilled water. The organic layer was dried over anhydrous Na2SO4 and concentrated to dryness under vacuum. The residue was chromatographed using a silica gel column to yield 1.68 g (87%) of pure 31, white powder. Mp 246-248 °C. MS (ESI-) m/z: 453.3 (M- - H) for C30H46O3. 1H NMR (300 MHz, CDCl3): δ 4.72, 4.58 (1H each, s, H-29), 3.09 (1H, m, H-19), 2.41-2.26 (2H, m, H-2), 1.69 (3H, s, H-30), 0.98, 0.97, 0.96, 0.92, 0.89 (3H each, s, 5 × CH3).

Methyl N-[3-deoxy-lup-20(29)-en-28-oyl]-leucinate (32)

A solution of 31 (500 mg, 1 eq), DMAP (0.6 eq) and EDCI (1.6 eq) in CH2Cl2 was stirred at 0 °C for 30 min. Leucine methyl ester (1.6 eq) and Et3N (1 eq) was then added into the system and stirred at room temperature overnight. The reaction was diluted with CH2Cl2 (20 mL) and washed with brine. The organic layer was then dried over anhydrous Na2SO4 and concentrated to dryness under vacuum. The residue was chromatographed using a silica gel column to yield 372 mg (58%) of pure 32, white amorphous powder. Mp 191-193 °C. MS (ESI+) m/z: 582.5 (M+ + H) for C37H59NO4NO4. 1H NMR (300 MHz, CDCl3): δ 5.87 (1H, d, J = 8.4 Hz, -CONH-), 4.70, 4.59 (1H each, s, H-29), 4.64 (1H, m, -NHCH-), 3.73 (3H, s, -COOCH3), 3.05 (1H, m, H-19), 2.41-2.26 (2H, m, H-2), 1.68 (3H, s, H-30), 1.06, 1.02 (3H each, s, leucine moiety -(CH3)2), 0.98 (3H, s, CH3), 0.96 (6H, s, 2 × CH3), 0.92, 0.89 (3H each, s, 2 × CH3).

Methyl N-[3-oxime-lup-20(29)-en-28-oyl]-leucinate (33)

A solution of 32 (230 mg, 1 eq), and hydroxylamine hydrochloride (4 eq) in pyridine (10 mL) was heated at 50 °C for 2 h. After cooling to room temperature, the reaction mixture was diluted with CH2Cl2 and washed three times by 20% HCl and brine. The organic layer was then dried over anhydrous Na2SO4 and concentrated to dryness under vacuum. The residue was chromatographed using a silica gel column to yield 235 mg (90%) of pure 33, white amorphous powder. Mp 213-215 °C. MS (ESI+) m/z: 582.5 (M+ + H) for C37H59NO4. 1H NMR (300 MHz, CDCl3): δ 5.87 (1H, d, J = 8.4 Hz, -CONH-), 4.70, 4.59 (1H each, s, H-29), 4.64 (1H, m, -NHCH-), 3.73 (3H, s, -COOCH3), 3.05 (1H, m, H-19), 2.20-2.15 (2H, m, H-2), 1.67 (3H, s, H-30), 1.05, 1.02 (3H each, s, leucine moiety -(CH3)2), 0.98 (3H, s, CH3), 0.96 (6H, s, 2 × CH3), 0.94, 0.92 (3H each, s, 2 × CH3).

Methyl N-[3β-amino-lup-20(29)-en-28-oyl]-leucinate (34)

To a solution of 33 (100 mg, 1 eq) and ammonium acetate (15 eq) in MeOH was added sodium cyanoborohydride (NaCNBH3, 20 eq) under nitrogen atmosphere. The reaction was cooled to 0 °C, and 15% aqueous titanium trichloride (TiCl3, 3 eq) was added dropwise over 45 min. The mixture was stirred at room temperature overnight, and then treated with 2 N NaOH until pH = 10. The solution was dried under vacuum and the resided aqueous solution was extracted with CH2Cl2 and washed with distilled water until pH = 7. The organic layer was then dried over anhydrous Na2SO4 and concentrated to dryness under vacuum. The residue was chromatographed using a silica gel column to yield 80 mg (82%) of pure 34, white amorphous powder. Mp 135-137 °C. MS (ESI+) m/z: 582.5 (M+ + H) for C37H59NO4. 1H NMR (300 MHz, CDCl3): δ 5.86 (1H, d, J = 7 Hz, -CONH-), 4.72, 4.58 (1H each, s, H-29), 4.64 (1H, m, -NHCH-), 3.73 (3H, s, -COOCH3), 3.05 (1H, m, H-19), 2.44 (1H, m, H-3), 2.10-1.90 (1H, m, H-13), 1.68 (3H, s, H-30), 0.97 (9H, s, CH3-23, leucine moiety-(CH3)2), 0.96, 0.94, 0.93, 0.92 (3H each, s, 4 × CH3).

Synthesis of BA-derivatives 30, 35, 39-40, 45-48

A solution of the appropriate BA analog (1 eq), DMAP (1.5 eq) and the appropriate acid anhydride (5 eq) in anhydrous pyridine (1.5 mL) was stirred at 160 °C for 2 h using microwave (Biotage). The reaction mixture was diluted with EtOAc (15 mL) and washed three times with 20% HCl solution and distilled water. The organic layer was dried over anhydrous Na2SO4 and concentrated to dryness under reduced pressure. The residue was chromatographed using a silica gel column to yield pure target compounds.

N-[3β-O-(3′,3′-Dimethylsuccinyl)-30-bromo-lup-20(29)-en-28-oyl]-leucine (30)

20 mg (32% yield) starting from 50 mg of 24; light yellow amorphous powder. Mp 105-107 °C. MS (ESI-) m/z: 774.5 (M- - H) for C42H66BrNO7. 1H NMR (300 MHz, CDCl3): δ 5.86 (1H, d, J = 8 Hz, -CONH-), 5.11, 5.02 (1H each, s, H-29), 4.65 (1H, m, -NHCH-), 4.54 (1H, dd, J = 11.2, 5.7 Hz, H-3), 3.90 (2H, s, H2-30), 2.99 (1H, m, H-19), 2.64-2.42 (2H, m, H-2′), 1.30, 1.26 (3H each, s, 2 × CH3-3′), 1.00 (6H, s, leucine moiety -(CH3)2), 0.96 (6H, s, 2 × CH3), 0.87, 0.86, 0.81 (3H each, s, 3 × CH3).

Methyl N’-[3β-N-(3′,3′-dimethylsuccinyl)-lup-20(29)-en-28-oyl]-leucinate (35)

37 mg (38% yield) starting from 80 mg of 34; white amorphous powder. Mp 187-189 °C. MS (ESI+) m/z: 711.5 (M+ + H), 733.4 (M+ + Na) for C43H70N2O6. 1H NMR (300 MHz, CDCl3): δ 5.69 (1H, br s, -CONH-), 4.70, 4.58 (1H each, s, H-29), 4.62 (1H, m, -NHCH-), 3.73 (3H, s, -COOCH3), 3.59 (1H, m, H-3), 2.99 (1H, m, H-19), 2.64-2.42 (2H, m, H-2′), 1.68 (3H, s, H-30), 1.30, 1.26 (3H each, s, 2 × CH3-3′), 1.09 (6H, s, leucine moiety -(CH3)2), 0.97 (6H, s, 2 × CH3), 0.92, 0.82, 0.80 (3H each, s, 3 × CH3).

N-[3β-O-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-4-piperidine butyric acid (39)

20 mg (41% yield) starting from 50 mg of 38, white amorphous powder. Mp 116-118 °C. MS (ESI+) m/z: 738.6 (M+ + H), (ESI-) m/z: 736.5 (M- - H) for C45H71NO7. 1H NMR (300 MHz, CDCl3): δ 4.72, 4.57 (1H each, s, H-29), 4.47 (1H, t, J = 7.5, H-3), 3.65-3.50 (4H, m, 28-CON(CH2CH2)2CH-), 2.98 (1H, m, H-19), 2.64-2.42 (2H, m, H-2′), 2.30 (2H, t, J = 7.2 Hz, -CH2COOH), 1.68 (3H, s, H-30), 1.27, 1.25 (3H each, s, 2 × CH3-3′), 0.95, 0.93, 0.84, 0.83, 0.82 (3H each, s, 5 × CH3). [α]25D -27.7 ° (c = 0.30, MeOH).

N-[3β-O-(4′,4′-Dimethylglutaryl)-lup-20(29)-en-28-oyl]-4-piperidine butyric acid (40)

12 mg (38% yield) starting from 30 mg of 38, white amorphous powder. Mp 143-145 °C. MS (ESI+) m/z: 752.4 (M+ + H), (ESI-) m/z: 750.4 (M- - H) for C46H73NO7. 1H NMR (300 MHz, CDCl3): δ 4.72, 4.57 (1H each, s, H-29), 4.47 (1H, t, J = 7.5, H-3), 3.65-3.50 (4H, m, 28-CON(CH2CH2)2CH-), 3.00 (1H, m, H-19), 2.35-2.30 (4H, m, H-2′, -CH2COOH), 1.68 (3H, s, H-30), 1.27, 1.25 (3H each, s, 2 × CH3-3′), 0.96 (6H, s, 2 × CH3), 0.89, 0.86, 0.82 (3H each, s, 3 × CH3). [α]25D -23.1 ° (c = 0.20, MeOH).

N’-[N-[3β-O-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]-aminomethane (45)

17 mg (48% yield) starting from 30 mg of 41, white amorphous powder. Mp 166-169 °C. MS (ESI+) m/z: 751.6 (M+ + H) for C46H74N2O6. 1H NMR (300 MHz, CDCl3): δ 5.43 (1H, br s, -CONHCH3), 4.69, 4.54 (1H each, s, H-29), 4.47 (1H, t, J = 7.5, H-3), 3.68-3.60 (4H, m, 28-CON(CH2CH2)2CH-), 2.98 (1H, m, H-19), 2.79 (3H, d, J = 3.4 Hz, -CONHCH3), 2.67-2.62 (2H, m, H-2′), 2.14 (2H, t, J = 6.8 Hz, -CH2CONHCH3), 1.65 (3H, s, H-30), 1.30, 1.25 (3H each, s, 2 × CH3-3′), 0.93, 0.91 (3H each, s, 2 × CH3), 0.90, 0.80, 0.79 (3H each, s, 3 × CH3). [α]25D -25.0 ° (c = 0.12, MeOH).

N’-[N-[3β-O-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]-morpholine (46)

23 mg (55% yield) starting from 35 mg of 42, off-white amorphous powder. Mp 122-124 °C. MS (ESI+) m/z: 807.6 (M+ + H) for C49H78N2O7. 1H NMR (300 MHz, CDCl3): δ 4.69, 4.54 (1H each, s, H-29), 4.45 (1H, t, J = 6.9, H-3), 3.66-3.61 (8H, m, 28-CON(CH2CH2)2CH-, -CON(CH2CH2)2O), 3.45-3.42 (4H, m, -CON(CH2CH2)2O), 2.99-2.82 (1H, m, H-19), 2.67-2.52 (2H, m, H-2′), 2.28 (2H, t, J = 7.8 Hz, -CH2CON(CH2CH2)2O), 1.65 (3H, s, H-30), 1.26 (6H, s, 2 × CH3- 3′), 0.92, 0.90 (3H each, s, 2 × CH3), 0.79 (6H, s, 2 × CH3), 0.77 (3H, s, CH3). [α]25D -19.1 ° (c = 0.41, MeOH).

N’-[N-[3β-O-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]-2-aminoethylmorpholine (47)

14 mg (41% yield) starting from 30 mg of 43, white amorphous powder. Mp 121-123 °C. MS (ESI+) m/z: 850.4 (M+ + H) for C51H83N3O7. 1H NMR (300 MHz, CDCl3): δ 7.01 (1H, br, s, -CONHCH2-), 4.70, 4.54 (1H each, s, H-29), 4.45 (1H, t, J = 10.2, H-3), 3.81-3.78 (8H, m, 28-CON(CH2CH2)2CH-, -CH2N(CH2CH2)2O), 3.48 (2H, m, -CONHCH2-), 2.85-2.70 (7H, m, -CH2N(CH2CH2)2O, H-19), 2.60-2.52 (2H, m, H-2′), 2.14 (2H, t, J = 7.5 Hz, -CH2CONHCH2-), 1.25 (6H, s, 2 × CH3-3′), 1.63 (3H, s, H-30), 0.93 (3H each, s, 2 × CH3), 0.90, 0.79, 0.72 (3H each, s, 3 × CH3). [α]25D -18.0 ° (c = 0.16, MeOH).

N’-[N-[3β-O-(3′,3′-Dimethylsuccinyl)-lup-20(29)-en-28-oyl]-4-piperidine-butanoyl]-3-aminopropylmorpholine (48)

17 mg (47% yield) starting from 30 mg of 44, white amorphous powder. Mp 125-127 °C. MS (ESI+) m/z: 864.6 (M+ + H) for C52H85N3O7. H NMR (300 MHz, CDCl3): δ 6.76 (1H, br, s, -CONHCH2-), 4.69, 4.54 (1H each, s, H-29), 4.30 (1H, m, H-3), 3.81-3.67 (8H, m, 28-CON(CH2CH2)2CH-, -CH2N(CH2CH2)2O), 3.28 (2H, m, -CONHCH2-), 2.96 (1H, m, H-19), 2.80-2.73 (6H, m, -CH2N(CH2CH2)2O), 2.60-2.52 (2H, m, H-2′), 2.16 (2H, t, J = 7.5 Hz, -CH2CONHCH2-), 1.65 (3H, s, H-30), 1.25, 1.24 (6H, s, 2 × CH3-3′), 0.92, 0.90 (3H each, s, 2 × CH3), 0.80, 0.79, 0.78 (3H each, s, 3 × CH3). [α]25D -14.1 °(c = 0.24, MeOH).

In Vitro Metabolic Stability Assessment

Materials

BA-derivatives 23 and 38 were synthesized and characterized in our study. NADPH, MgCl2, KH2PO4, formic acid and ammonium acetate were purchased from Sigma-Aldrich. Reference compounds (fast-metabolized: buspirone, propranolol; moderate-metabolized: atenolol; and slow-metabolized: imipramine) were also purchased from Sigma-Aldrich. HPLC-grade acetonitrile and water was purchased from VWR. Pooled human liver microsomes (Lot No# 70196) were purchased from BD biosciences (Woburn, MA).

Sample Preparation

Stock solutions of 23 and 38 (1 mg/mL) were prepared by dissolving the pure compound in methanol and stored at 4 °C. For measurement of metabolic stability, four reference compounds as well as test compounds 23 and 38 were brought to a final concentration of 3 μM with 0.1 M potassium phosphate buffer at pH 7.4, which contained 0.2 mg/mL human liver microsome and 5 mM MgCl2. The incubation volumes were 800 μL. Reactions were started by adding 80 μL of NADPH (final concentration of 1.0 mM) and stopped by taking the aliquots over time, then adding to 1.5 volumes of ice-cold acetonitrile. Incubations of all samples were conducted in duplicate. For each sample, 100 μL aliquots were taken out at 0, 5, 15, 30, 60, 120 min time points. After addition of 150 μL ice-cold acetonitrile, the mixture was centrifuged at 12,000 rpm for 5 min at 0°C. The supernatant was collected and 20 μL of the supernatant was directly injected to LCMS. The following controls were also conducted: 1) positive control incubations that contain liver microsomes, NADPH and the fast-metabolized substrate propranolol; 2) negative control incubations that omit NADPH; and 3) baseline control that only contain liver microsomes and NADPH.

HPLC-MS Conditions

Analysis was carried out on Shimadzu LCMS-20 with an electrospray ionization source (ESI). An Alltima C18 5 μm 150 mm × 2.1 mm column was used with a gradient elution at a flow rate of 1.5 mL/min. The initial elution condition was acetonitrile (B) in water (A, with 0.1% formic acid and 5 mM ammonium acetate) at 55%. After staying at initial condition for 3 min, the concentration of B increased linearly to 90% at 15 min, and stayed at 90% for 2 min. The mobile phase was then returned to the initial condition and re-equilibrated for 3 min. The MS conditions were optimized to detector voltage: +1.35 kV, acquisition mode: SIM of the appropriate molecular weights of the testing compounds. The CDL temperature is 200 °C, heat block is 230 °C and neutralizing gas flow is 1.5 L/min. Samples were injected by auto-sampler. Electrospray ionization was operated in the positive ion mode. Full-scan spectra were also monitored over the range of 180 - 1000 m/z.

HIV-1IIIB Replication Inhibition Assay in MT-2 Lymphocytes

The evaluation of HIV-1 inhibition was carried out as follows. The human T-cell line, MT-2, was maintained in continuous culture with complete medium (RPMI 1640 with 10% fetal calf serum supplemented with L-glutamine at 5% CO2 and 37 °C. Test samples were first dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL to generate master stocks with dilutions made into tissue culture media to generate working stocks. The following drug concentrations were routinely used for screening: 100, 20, 4 and 0.8 μg/mL. For agents found to be active, additional dilutions were prepared for subsequent testing so that an accurate EC50 value could be determined. Test samples were prepared, and to each sample well, was added 90 μL of media containing MT-2 cells at 3×105 cells/mL and 45 μL of virus inoculum (HIV-1 IIIB isolate) containing 125 TCID50. Control wells containing virus and cells only (no drug) and cells only (no virus or drug) were also prepared. A second identical set of samples were added to cells under the same conditions without virus (mock infection) for cytotoxicity determinations (CC50 defined below). In addition, AZT and bevirimat were also assayed during each experiment as positive drug controls. On day 4 PI, the assay was terminated and culture supernatants were harvested for p24 antigen ELISA analysis. The compound cytotoxicity was determined by XTT using the mock-infected sample wells. The detailed procedure was described previously.33,34 If a test sample inhibited virus replication and was not cytotoxic, its effects were reported in the following terms: EC50, the concentration of the test sample that was able to suppress HIV replication by 50%; CC50, the concentration of test sample that was toxic to 50% of the mock-infected cells; and therapeutic index (TI), the ratio of the CC50 to EC50.

HIV-1NL4-3 Replication Inhibition Assay in MT-4 Lymphocytes

A previously described HIV-1 infectivity assay was used.30,35 A 96-well microtiter plate was used to set up the HIV-1NL4-3 replication screening assay. NL4-3 variants at a multiplicity of infection (MOI) of 0.01 were used to infect MT4 cells. Culture supernatants were collected on day 4 post-infection for the p24 antigen capture using an ELISA kit from ZeptoMetrix Corporation (Buffalo, New York).

Supplementary Material

1_si_001

Acknowledgement

This investigation was supported by Grant AI-077417 from the National Institute of Allergy and Infectious Diseases (NIAID) awarded to K.H.L.

Abbreviations

AIDS
acquired immunodeficiency syndrome
HIV-1
human immunodeficiency virus type 1
HAART
highly active antiretroviral therapy
NRTIs
nucleoside/nucleotide reverse transcriptase inhibitors
NNRTIs
non-nucleoside reverse transcriptase inhibitors
PIs
protease inhibitors
MI
maturation inhibitor
BA
betulinic acid
P24 (CA)
capsid
Bevirimat (DSB, PA-457)
3′,3′-dimethylsuccinyl-betulinic acid
RPR103611
(3S,4S)-N’-[N-[3β-hydroxylup-20(29)-en-28-oyl]-8-aminooctanoyl]-4-amino-3-hydroxy-6-methylheptanoic acid
IC9564
(3R,4S)-N’-[N-[3β-hydroxylup-20(29)-en-28-oyl]-8-aminooctanoyl]-4-amino-3-hydroxy-6-methylheptanoic acid
A43-D
[[N-[3β-O-(3′,3′-dimethylsuccinyl)-lup-20(29)-en-28-oyl]-7-aminoheptyl]-carbamoyl]methane
CYPs
cytochrome P450;
UGTs
UDP-glucuronosyltransferases
FMO
flavin containing monooxygenase
NBS
N-bromosuccinimide
PDC
pyridinium dichromate
HOBt
hydroxybenzotriazole
EDCI
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
DMAP
4-(dimethylamino)pyridine
AZT
zidovudine

Footnotes

Anti-AIDS Agents 78. For part 77, see S. Xu, X. Yan, Y. Chen, P. Xia, D. Yu, K. Qian, Y. Xia, Z.Y. Yang, S.L. Morris-Natschke, and K.H. Lee, “Anti-AIDS Agents 77. Synthesis and Anti-HIV Activity of 2′-Monomethyl-4-methyl DCK and 1′-Thia-4-methyl DCK Analogs”, J. Med. Chem., (submitted).

Correspondence to: Kuo-Hsiung Lee, Natural Products Research Laboratories, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599. E-mail: .ude.cnu@eelhk

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