Ahmpatinin iBu, a new HIV-1 protease inhibitor, from Streptomyces sp. CPCC 202950

Ahmpatinin iBu (1) and statinin iBu (2), two new linear peptides, a novel pyrrolidine derivative, (−)-(S)-2-[3-(6-methylheptanamido)-2-oxopyrrolidin-1-yl] acetic acid (3), and three known pepstatin derivatives (4–6) along with their corresponding methanolysis artifacts (7–9) were isolated from Streptomyces sp. CPCC 202950. Their structures were elucidated on the basis of extensive spectroscopic data using Marfey's analysis, chiral-phase HPLC, and ECD and OR calculation to determine the absolute configurations. Compound 1 contains an unusual amino acid, 4-amino-3-hydroxy-5-(4-methoxyphenyl)pentanoic acid (Ahmppa), and 3 is the first natural product with a 2-(3-amino-2-oxopyrrolidin-1-yl)acetic acid system. Compounds 1, 2, and 4–9 are HIV-1 protease inhibitors. In particular, ahmpatinin iBu (1) exhibits significant inhibitory activity against HIV-1 protease with an IC50 value of 1.79 nM. A preliminary structure–activity relationship is discussed.


Introduction
The most effective treatment for HIV infection, HAART (Highly Active AntiRetroviral Therapy) that including several HIV-1 protease inhibitors combination with reverse transcriptase inhibitors, and other inhibitors with integrase, membrane fusion, and viral attachment, have signicantly reduced the mortality of AIDS patients and improved the quality of life of those infected with HIV. [1][2][3][4] However, according to World Health Organization and Joint United Nations Program on HIV/AIDS data, in 2016, there were still 36.7 million people living with HIV, 1.8 million newly infected and 1.0 million HIV-related deaths. 5,6 Therefore, AIDS is still a substantial threat to global public health. HIV-1 protease (HIV-1 PR) is an essential enzyme in the life cycle of HIV and has been used as a promising target for AIDS therapy. [7][8][9] Although 11 HIV-1 PR inhibitors such as amprenavir, indinavir, nelnavir, ritonavir, saquinavir, and tipranavir have been approved by the FDA and applied extensively in clinical evaluations, 10,11 drug toxicity, resistance, and drug-drug interactions remain serious problems for HIV/AIDS treatments, 1,12-14 prompting us to discover new anti-HIV drugs.
In addition to compounds 1-3, the known pepstatin Ac (4), 18,22 pepstatin Pr (5), 18,22 pepsinostreptin (6), 18 pepstatin Ac methyl ester (7), pepstatin Pr methyl ester (8), and pepsinostreptin methyl ester (9) were also isolated and identied from the strain Streptomyces sp. CPCC 202950. Compounds 4-6 incubated with methanol showed the presence of corresponding methyl esters 7-9, respectively. These results indicated that 7-9 were indeed artifacts derived from methanolysis during the isolation process. Furthermore, a new benzamide analog and seven known compounds were isolated from the inactive fractions of the crude extract in our previous report. 23 Pepstatin and ahpatinin derivatives have been shown to exhibit signicant inhibitory activity against aspartic protease. 18,24-28 HIV-1 PR is a 22 kDa dimeric aspartyl protease. As expected, compounds 1 and 2 along with pepstatin derivatives 4-9 from the active fractions showed signicant inhibitory activity against HIV-1 protease with IC 50 values of 1.79 nM to 3.19 mM, while compound 3 was inactive (the positive control indinavir gave an IC 50 value of 1.82 nM) in preliminary in vitro assays (Table 3). A comparison of the activities of 4-6 and 7-9 demonstrated that the exposed terminal carboxyl group was very important for the HIV-1 protease inhibitory activity. Analysis of the inhibitory abilities of 1 and 4-9 indicated that the Ahmppa unit was better than the statine moiety. Compound 2 showed weaker inhibitory ability than 1 and 4-9, suggesting that the presence of the statine or Ahmppa unit could enhance the HIV-1 protease inhibitory activity. In addition, all of the compounds were not active at toward Hela, HepG2, and U2OS tumor cells at 100 mM in in vitro tests.

General experimental methods
ORs were measured using a Rudolph Research Autopol III automatic polarimeter. UV, CD, and IR spectra were recorded using a Cary 300 spectrometer, a JASCO J-815 CD spectrometer, and a Nicolet 5700 FT-IR spectrometer (FT-IR microscope transmission), respectively. 1 H and 13 C NMR spectra were obtained at 600 and 150 MHz, respectively, using a Bruker-AVIIIHD-600 spectrometer with solvent peaks used as references. HR-ESIMS data were measured using a Micromass Autospec-Ultima ETOF spectrometer and Thermo LTQ Orbitrap XL mass spectrometer. Column chromatography was performed using silica gel (200-300 mesh, Qingdao Marine Chemical Inc., China) and MCI gel CHP20P (Mitsubishi Chemical Corporation, Tokyo, Japan). HPLC separation was performed using an Agilent 1200 series (quaternary pump, autosampler, diode array detector) with a Shiseido Capcell-Pak C 18 MGII column (5 mm, 250 Â 10 mm). TLC was performed using glass-precoated silica gel GF254 plates (Qingdao Marine Chemical Inc., Qingdao, China). Cervical cancer (Hela), hepatocellular carcinoma (HepG2), and human osteosarcoma (U2OS) cell lines were obtained from the National Infrastructure of Cell Line Resource (Beijing, China).

Microorganism and fermentation
Strain CPCC 202950 was identied as a member of the genus Streptomyces on the basis of 16S rRNA sequence analysis and deposited at the China Pharmaceutical Culture Collection (Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, no. CPCC 202950).
The strain was cultured on slants of YM (0.4% yeast extract, 1% malt extract, 0.4% glucose, and 1.2% agar) at 28 C for 7 d. Agar plugs were cut into small pieces (about 0.5 Â 0.5 Â 0.5 cm 3 ) under aseptic conditions and 5 of these pieces were used to inoculate in three Erlenmeyer asks (500 ml), each containing 100 ml of media (glucose 0.5%, yeast extract 0.5%, soluble starch 2.0%, soybean meal 1%, peptone 0.5%, beef extract 0.5%, corn steep liquor 0.4%, CaCO 3 0.4%, and CoCl 2 -$6H 2 O 0.002%), and the nal pH was adjusted to 7.2. Aer Fig. 4 Experimental ECD spectrum of 3 (blue) and the calculated ECD spectra of (R)-3 (red) and (S)-3 (black). sterilization, three asks of the inoculated media were incubated at 28 C on a rotary shaker at 220 rpm for two days to prepare the seed culture. Spore inoculum was prepared by suspending the seed culture in sterile, distilled H 2 O to give a nal spore/cell suspension of 1 Â 10 6 /ml. Fermentation was carried out in 30 Fernbach asks (500 ml), each containing 80 g of rice. Distilled H 2 O (120 ml) was added to each ask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. Aer cooling to room temperature, each ask was inoculated with 10.0 ml of the spore inoculum and incubated at 28 C for 30 day.

Extraction and isolation
The fermented material was ultrasonicated with 95% EtOH (2 Â 8.0 l Â 40 min), and the EtOH extracts were combined and evaporated under reduced pressure to yield an aqueous suspension (2.0 l). The suspension was partitioned with EtOAc (4 Â 2.0 l). The EtOAc extract (15.0 g) was chromatographed over a silica column using a gradient elution of increasing MeOH (0-100%) in CH 2 Cl 2 to give 10 fractions (F 1 -F 10 ). The fraction of F 6 was also subjected to silica gel CC using a gradient elution of increasing MeOH (0-100%) in CH 2 Cl 2 to give six parts (F 6-1 - ECD and OR calculations of compound 3. Conformational analysis of the S-enantiomer was carried out via Monte Carlo searching with the MMFF94s molecular mechanics force eld using the Spartan 10 soware. 31 Seventy-two geometries having relative energies within 6 kcal mol À1 were optimized using DFT at the B3LYP/6-31G (d) level in vacuum with the Gaussian 09 program. 32 The 52 B3LYP/6-31G(d)-optimized conformers with relative energies ranging from 0 to 4.0 kcal mol À1 were then reoptimized at the wB97XD/DGDZVP level in acetonitrile. ECD and OR computations for all wB97XD/DGDZVP-optimized conformers were carried out at the CAM-B3LYP/DGDZVP and B3LYP/DGDZVP levels in acetonitrile, respectively. Boltzmann statistics were computed for ECD simulations with a standard deviation of s 0.3 eV. The ECD spectra were then simulated using the GaussSum 2.25 program. 33 The nal ECD spectrum of (S)-3 was obtained according to the Boltzmann distribution theory and the relative Gibbs free energy (DG).
Analysis of the inhibition of HIV-1 protease activity. This was measured using the previously described protocol. 15 The positive control indinavir gave an IC 50 value of 1.82 nM.
Cytotoxic activity assay. This was measured using the previously described protocol. 34 The positive control doxorubicin showed in vitro cytotoxicity against cervical cancer (Hela), hepatocellular carcinoma (HepG2), and human osteosarcoma (U2OS) cell lines with IC 50 values of 1.42, 1.56, and 2.78 mM, respectively.

Conflicts of interest
There are no conicts to declare.