Human cancer inhibitory pentapeptide amides and esters

【0001】

[Technical field to which the invention belongs]

The present invention generally relates to chemotherapy for cancer, and more particularly to the structuring and synthesis of peptideamides and ester derivatives of drastatin 10 which are specific tumor inhibitors useful in chemotherapy.

【0002】

[Background of Invention and Conventional Technology]

The ancient marine invertebrate species Phyla Bryozoa, Molluska and Porifera have existed in the ocean for billions of years. As a result of trillions of evolutionary chemical biosynthetic reactions, such organisms have reached current levels of tissue, regulation and defense.

【0003】

However, marine sponges have changed their physical characteristics very little over the last 500 million years. This suggests that this animal has extremely effective chemical resistance to evolution in response to changes in environmental conditions during that period. Recognition of the potential to utilize this biologically active marine animal for medical purposes was recorded in Egypt around 2700 BC, and by 200 BC some species of Aplysia (Sea hare). ) Extract was used in Greece as having a therapeutic effect. This, along with the observation that marine animals such as invertebrates and sharks produce few cancers, has paved the way for systematic studies of anticancer compounds in marine animals and plants.

【0004】

By 1968, certain marine products could provide novel anti-neoplasms and / or cytotoxic agents useful in chemotherapy and could also lead to effective compounds for the control and / or treatment of viral diseases. Sufficient evidence was available based on experimental cancer studies at the National Cancer Institute (NCI) in the United States.

【0005】

These marine products were also believed to have potential useful drug candidates with unexpected structures not found elsewhere in medical chemistry. Fortunately, these expectations have come true, for example, with the discovery of bryostatins, dorastatins and cephalotatins. Many of them are currently undergoing preclinical development or clinical research in humans.

【0006】

Current medical chemistry researchers are well aware of the time lag between the isolation of new compounds and their launch. This often takes years and can be decades or more. As a result, industry has developed a dual-purpose testing standard system in connection with the US Government. One is the withdrawal of substances that have been tested and found to be economically unprofitable to continue. The other is to identify compounds that have shown high potential for success and thus have the benefits of continued research and development, as well as guarantee the costs required to meet the stringent legal requirements that control the ultimate market position. It is an even more important purpose that helps to do so.

【0007】

The current cost of developing the data needed to legally market new pharmaceutical compounds is as high as $ 10 million per compound. Economics dictates that such huge investments should only be made when there is a reasonable recovery potential. No investment is made when that is not possible, and without it, the requirements for research to discover these potential life-saving compounds disappear.

【0008】

Current studies of cancer control in the United States are being coordinated by the National Cancer Institute (NCI). The NCI has established a systematic protocol to determine if a substance has anti-cancer properties. This protocol, which includes testing the substance against a standard cell line panel containing 60 human tumor cell lines, has been proven and approved in the scientific field. Established statistical methods for the analysis of the results obtained in this protocol and standardized test are described in detail in the literature. See below for a detailed description of the test protocol. Boyd, Dr. Michael R., Principles & Practice of Oncology, PPO Updates, Vol.3, No.10, October 1989. See below for statistical analysis methods. Paull, KD, Display and Analysis of Patterns of Differential Activity of Drugs Against Human Tumor Cell Lines; Development of Mean Graph and COMPARE Algorithm , Journal of the National Cancer Institute Reports, Vol.81, No.14, Page 1088, July 14,1989. All of these references are incorporated herein by this reference.

【0009】

Numerous substances have been discovered that exhibit prominent anti-neoplasmic or tumor-inhibiting properties. As mentioned above, many of these compounds have been extracted from marine animals such as sponges and Aplysia, with great difficulty. Once these compounds have been isolated and tested, the practical question of how to produce the commercial requirement of the desired substance arises.

【0010】

Quinine, which is obtained in practical amounts from the bark of the kina plant, is different from compounds that are extracts of marine products. The collection and treatment of these latter compounds from natural sources is extremely impractical or completely impossible. Ignoring the ecological consequences, the number of these organisms and the cost of collection and extraction make the work impossible. Artificial synthesis of active compounds is the only possible solution.

【0011】

Therefore, it is essential to determine the structure of these anti-neoplastic compounds. Once the structure is determined, then the synthetic means must be determined. This is often long and difficult due to the specific complexity of these naturally occurring evolutionarily modified compounds. In addition, it is necessary to study which parts of the natural compound have the desired properties so that the simplest structure with the desired properties can be focused on.

[Means for solving problems]

【0012】

Isolation, structure determination and synthetic regeneration of potentially useful anti-neoplastic peptides provide the most promising approaches to new anti-cancer agents. Continued research along these lines has led to the discovery and synthetic regeneration of five effective novel anti-cancer peptides. Natural and some modified amino acids were used in the synthesis of these peptides. The modified amino acids selected herein are components of known Drastatin 10 and Drastatin 15, which are well-known structural peptides with significant anti-neoplastic activity. Currently, Drastatin 10 is the most important of the Drastatins and is a potentially useful anticancer drug. Novel compounds with significant activity against a range of human cancer cell lines are described herein. The structures of these compounds, their reference numbers, and synthetic charts are shown below.

【0013】

[Chemical 4]<img he="160" id="000002" wi="159" file="2_0003579751.tif" img-format="tif" img-content="drawing" /><img he="189" id="000003" wi="159" file="3_0003579751.tif" img-format="tif" img-content="drawing" /><img he="121" id="000004" wi="159" file="4_0003579751.tif" img-format="tif" img-content="drawing" /> 【0014】

The novel peptides disclosed herein are obtained by introducing a peptide bond between the selected amino acid and the modified amino acid and binding the obtained di and try peptide to obtain a novel peptide having extremely high anticancer activity. It is composed. For convenience, these compounds shall be represented by the reference numbers used in the above flowchart. This disclosure includes the identification and synthesis of five novel compounds: two pentapeptides (12 and 14), two tetrapeptide alkyl esters (16a-b) and one tetrapeptide amide (16c). ..

【0015】

The synthesis of these five compounds was specifically achieved using the following methods.

【0016】

A common peptide (5) is required for the synthesis of two pentapeptides (12 and 14). Tripeptide (5) is synthesized starting from the modified amino acid dry isoleucine (Dil). Drysoloicine is combined with N-cbz- (L) -isoleucine (1) in the presence of diisopropylethylamine using BrOP as a coupling agent to dipeptide NZ-Ile-Dil-OBu.^tGet (3). The N-carbobenzyloxy protecting group of dipeptide (3) was then removed in silochexane with 10% Pd-C to give it a free base, which was then dravaline (Dov; modified amino acid) using diethylcyanophosphate as a coupling agent. Dov-Ile-Dil-OBu, which is the desired tripeptide in combination with^t(5). In a similar manner, three alkyl groups of different lengths, namely ventil, octyl and hexyl groups, are selected for the synthesis of the tetrapeptide alkyl ester / amide (16). The desired t-boc-draploin ester (6; modified amino acid) is readily synthesized by reaction with the corresponding alkyliodides (7a and 7b) in the presence of sodium bicarbonate (in dry dimethylformamide). To. The t-boc doraproinamide (8c) is produced by reacting t-boc-draploin with hexylamine (7c) in the presence of diethylcyanophosphate and triethylamine. The t-boc protecting group of trypeptide (5) and t-boc-dap ester / amide (8a-c) is then removed with trifluoroacetic acid to give the corresponding tfa salt (9,10a-c).

【0017】

The resulting tripeptide-tfa salt (9) binds to two known salts, namely tfa * Dap-Doe (11) and tfa * Dap-Met-OMe (13). These bonds are carried out with DECP to give the pentapeptides (12 and 14) in good yield. Similarly, the tfa salt of dap-ester / amide (10a-c) is combined with the tripeptide-tfa salt and tfa * Dovl-Val-Dil-COOH (15) using EDCP as a coupling agent to obtain a good yield of tetrapeptide. Obtain an ester / amide (16a-c).

【0018】

All of these compounds exhibit excellent growth inhibitory effects when administered to various human cancer and mouse leukemia cell lines. The biological results are disclosed in Tables 1 and 2 below.

【0019】

Therefore, the first object of the present invention is the identification and synthesis of a novel peptide derivative of drastatin 10 which shows a remarkable inhibition of cell growth activity. Another object of the present invention is to isolate the active site of a drastatin 10 derivative that can be added to other structures that create novel compounds that show significant growth inhibition when measured in various human cancer and mouse leukemic cell lines. It is to be.

【0020】

These and other purposes, such as those shown below, are readily met by the present invention in a highly unexpected manner, as is readily apparent from the detailed description of the exemplary embodiments below.

Description of Preferred Embodiments

【0021】

In vitro studies are an absolute essential factor in advancing projects to find new compounds used to combat cancer destruction. Without such screening, the method of obtaining new candidate compounds would be extremely complex, if not impossible, and expensive. In order to understand this method and to recognize the results exhibited by some of the substances disclosed herein, one must first understand the method, naming, and data analysis used. The terms used are briefly explained below.

【0022】

ED₅₀(P388) and GI₅₀(HTCL) represents the dose of drug that reduces tumor / cell growth by 50%. ED₅₀And GI₅₀There is no mathematical difference between them, and both are calculated by the same formula. The only difference is the historical usage.

【0023】

TGI stands for Total Growth Inhibition and represents the dose of drug required to achieve zero% growth (ie, at the end of the experiment, having exactly the same number of cells as at the start of the experiment). It is indistinguishable whether the same number of cells that were produced died (equilibrium) or did not grow (complete inhibition).

【0024】

LC₅₀Is an abbreviation for Lethal Concentration 50% (50% lethal concentration), which represents the concentration of a drug that reduces the number of cells present at the start of the experiment by half.

【0025】

Each drug is tested at a 5-step dose of 100-10-1-0.1-0.01 μg / ml. Calculate% growth for each dose. 2 (or 3) doses of growth values greater than or equal to 50% and less than or equal to (or approximate) ED using linear regression calculation₅₀/ GI₅₀Used to calculate the value. If neither dose shows a growth value of 50% or less, the result is ED₅₀&gt; Expressed as (highest value). If neither dose is higher than 50% growth, ED₅₀Expressed as &lt;(minimum value). Similar calculations include TGI at 0% growth and LC at -50% growth.₅₀To do.

【0026】

At the start of each experiment, cells from in vitro cell culture are transplanted into a suitable tube or microquantity plate. Immediately count a set of control tubes / plates to determine the number of cells at the start of the experiment. This is a "basic count" or a "T zero count". At the end of the experiment (48 hours later), a second set of control tubes / plates is analyzed to determine the "control growth" value. Cell growth (or death) relative to the initial cell number is used to determine the "growth percentage".

【0027】<img he="67" id="000005" wi="131" file="5_0003579751.tif" img-format="tif" img-content="drawing" /> 【0028】

Having disclosed appropriate definitions and data analysis techniques above, this disclosure can be used for the particular compounds disclosed herein.

【0029】

The synthesis of potentially useful peptides provides the most essential and promising approach to new types of anti-cancer and immunosuppressive agents. Drastatins, an unprecedented chain and ring anti-neoplasm and / or cytotoxic peptide (obtained from the Aplysia Dolabella auricuIaria in the Indian Ocean), provide an excellent lead to synthetic modification. The extremely large amount of Aplysia produces a large number of structurally specific peptides with excellent anti-neoplastic activity. The chain pentapeptide, drastatin 10, is currently the most important and potentially useful anti-neoplasmic agent. Drastatin 10 shows the best anti-neoplastic activity profile for various currently known cancer screens. Recently, the total synthesis and absolute configuration of this structurally unique and biologically active peptide have been found. This compound was tested non-vipo and showed significant activity as shown below.

【0030】

Experimental anticancer (T / C; μg / kg) P388 lymphocyte leukemia toxic (13.0) 155 and 17% cure (6.5) 146 and 17% cure (3.25) 137 (1.63) L1210 Lymphocyte Leukemia 152 (13) 135 (6.5) 139 (3.25) 120 (1.63) B16 Melanoma 238 and 40% cure (11.11) 182 (6.67) 205 (4.0) 171 (3.4) 142 (1.44) M5076 Ovarian sarcoma toxic (26) 166 (13) 142 (6.5) 151 (3.25) LOX human melanoma xenograph (nude mouse) toxic (52) 301 and 67% cure (26) 301 and 50% cure (13) 206 and 33% cure (6.5) 170 and 17% cure (3.25) LOX340 and 50% cure in another experiment (43) 181 and 30% cure (26) 192 (15) 138 and 17% cure (9.0) Human Breast xenograph (nude mouse) toxic (26) 137 (13) 178 (625) OVCAR-3 Human Ovary Xenobraf (Nude Mouse) 300 (40) MX-1 Human Breast Xenograph (Tumor Suppression) 14 (52) 50 (26) 61 (13) 69 (6.25) [0031]

Drastatin 10 was also tested against a minipanel from the NCI primary screen. The results are as follows, GI for the following cell lines₅₀The amount of drastatin 10 required to obtain (μg / ml) is shown. OVCR-3 (A) 9.5 × 10^-7SF 295 (B) 7.6 × 10^-8A498 (C) 2.6 × 10^-5NCl-H460 (D) 3.4 × 10^-6KM20L2 (E) 4.7 × 10^-6SK-MEL-5 (F) 7.4 × 10^-6Similarly, compounds 12, 14, 16a, 16b and 16c of the present invention were also tested on the NCI in vitro minipanel. GI for each of the 6 cell lines₅₀, TGI and LC₅₀Was calculated for each compound. Each compound was also tested on the PS-388 cell line and ED₅₀Was calculated. The NCI minipanel protocol was created by Dr. MR Boyd and is known to those of skill in the art, except for the number of cell lines. The test method for PS-388 leukemia is the same as in the alternative NCI-P-388 screening test, which is also known to those of skill in the art.

【0032】

[table 1]<img he="16" id="000006" wi="159" file="6_0003579751.tif" img-format="tif" img-content="drawing" /><img he="182" id="000007" wi="159" file="7_0003579751.tif" img-format="tif" img-content="drawing" /> 【0033】

[Table 2]<img he="27" id="000008" wi="154" file="8_0003579751.tif" img-format="tif" img-content="drawing" /><img he="167" id="000009" wi="159" file="9_0003579751.tif" img-format="tif" img-content="drawing" /> 【0034】

General Law AN-Z-Ile-Dil-OBu^tThe synthesis of (3) is performed as follows. Diisopropylethylamine in a solution of dry isoleucine t-butyl ester hydrochloride (2; 1 mM) and NZ- (L) -isoleucine (1; 1.1 mM) in dichloromethane (10 mL) cooled to ice bath temperature (0-5 ° C). (3 mM) Then BrOP (2 mM) is added and the resulting solution is stirred at the same temperature for 2 hours. The solvent is removed under reduced pressure and the residue is chromatographed on a silica gel column using 1: 3 acetone-hexane as the solvent to give the desired dipeptide as an oil (3; 40%). Rf0.34 (1: 4 Acetone-, Hexane); [α]_D²⁵-7.5 ° (c1.19, CHCl₃); IR (neat): 3393,3374,3295,2967,2934,2878,1724,1638,1528,1501,1456,1412,1283,1368,1296,1250,1229,1153,1099,1038,1028,9890 , 959,845,777,739,698 and 619 cm^-1;¹H-NMR (CDCl₃, 300MHz): 7.25 (m, 5H, ArH), 5.37 (d, J = 9.5Hz, 1H, NH), 4.99 (s, 2H, ArCH)₂), 4.60 (m, 1H, dilN-CH), 4.43 (dd, J = 6.8 and 9.5Hz, 1H, IleCαH), 3.79 (m, 1H, CH-OMe), 3.24 (s, 3H, OMe), 2.34 (brd, J = 15.5Hz, 1H, HCH-CO), 2.20 (dd, J-9.3 and 15.5Hz, 1H, HCH-CO), 1.50-0.9 (m, 6H, 2 × CH₂, 2 × CH), 1.35 (s, 9H, t-Bu), 0.88 (d, J = 8.1Hz, 3H, CH-CH₃), 0.86 (d, J = 7.2Hz, 3H, CH-CH₃), 0.78 (t, J = 7.4Hz, 3H, CH₂-CH₃) And 0.73 (t, J = 7.6Hz, 3H, CH₂CH₃EIMS (m / z): 506 (M⁺, 0.1), 433 (0.8), 393 (0.7), 347 (12), 279 (2), 276 (3), 248 (1), 239 (2), 236 (1), 230 (1), 220 (6), 190 (4), 186 (6), 177 (3), 176 (18), 172 (3), 171 (1), 155 (2), 154 (6), 146 (9), 143 (3), 141 (1), 130 (1), 128 (4), 108 (4), 107 (4), 103 (6), 101 (10), 100 (100), 99 (2), 98 (2), 97 (1), 96 (1), 95 (1), 92 (9), 91 (78) and 57 (18%).

【0036】

General Law BDov-Ile-Dil-OBu^tThe synthesis of (5) is performed as follows. Z-Ile-Dil-OBu^tA solution of (3; 0.2 mM) is dissolved in anhydrous methanol (2 mL) and cyclohexane (2 mL) is added in an argon atmosphere. 10% Pd-C (0.05 g) is added to this solution and the mixture is heated to reflux for 10-15 minutes. The catalyst is removed by filtration through a layer of Celite, the solvent is removed under reduced pressure and the residue is dried under high vacuum for 2 hours.

【0037】

Trimethylamine (0.8 mM) in a dry dichloromethane (2 mL) solution of the above free base and N, N-dimethyl- (L) -valine (4; 0.2 mM), then DECP (0.22 mM) in an argon atmosphere from 0 to Add at 5 ° C. After stirring at the same temperature for 2 hours, the solvent is removed and the residue is chromatographed on a silica gel column with 1: 3 acetone-hexane as the solvent to give the desired tripeptide t-butyl ester as a colorless solid (5; 65%); Melting point 64 ~ 65 ° C; Rf0.27 (1: 4 Acetone-Hexane); [α]_D²⁵-40 ° (c0.12, CHCl₃); IR (thin film): 3302,1967,2934,2878,1732,1661,1622,1485,1462,1454,1416,1383,1368,1300,1283,1258,1200,1153,1101,1037 and 619cm^-1;¹N-NMR (CDCl₃, 300MHz): 6.78 (d, J = 8.7Hz, 1H, NH), 4.79 (dd, J = 7.2 and 9.3Hz, 1H, IleCα-H), 4.7 (m, 1H, dilCHN), 3.86 (m, 1H) , CH-OMe), 3.33 (s, 3H, OMe), 2.99 (s, 3H, dilN-Me), 2.2-2.5 (m, 2H, CH₂-CO), 2.21 (s, 6H, NMe₂), 2.05 (m, 1H, dovCα-H), 1.2-1.8 (m, 7H, 2 × CH₂, 3 × CH), 1.43, 1.54 (s, 9H, t-Bu) and 0.75-0.99 (m, 18H, 6 × CH)₃); EIMS (m / z): 4.99 (M⁺, 0.3), 4.56 (0.6), 241 (3), 186 (1), 128 (1), 125 (1), 103 (2), 101 (10), 100 (100) .99 (1), 98 (1), 91 (2), 86 (2), 85 (3), 84 (2) and 57 (8%).

【0038】

General method The synthesis of Ct-Boc-draproin ester / amides is carried out as follows. Alkyliodide (7; 1.2 mM) and sodium bicarbonate (2 mM) are added to a solution of t-Boc-draproin (6; 1 mM) in dry dimethylformamide (5 mL), and the resulting solution is stirred at room temperature for 24 hours. Dichloromethane (50 mL) is added, the organic layer is washed with water (2 x 25 mL) and dried. The residue from which the solvent has been removed under reduced pressure is chromatographed on a silica gel lamb in an appropriate solvent system to obtain the desired ester.

【0039】

General Method D Triethylamine (2 mM) and diethylcyanophosphate (1.1 mM) in a solution of t-Boc-draploin (6; 1 mM) and amine (7c) in dry dichloromethane (5 mL) cooled to ice-cooled temperature under an argon atmosphere. Add. The resulting solution is stirred at the same temperature for 1.5 hours. The solvent is removed under reduced pressure and the residue is chromatographed on a silica gel column in a suitable solvent system to give the desired amide.

【0040】

The t-Boc-Dap pentyl ester (8a) is synthesized as follows. Reaction of t-Boc-draproin (6) with pentyliodide (7a) according to General Method C, the resulting residue was produced on a silica gel column using 1: 3 acetone-hexane as an eluent to make the desired octyl ester colorless. Obtained as a liquid (8a, 50%). Rf0.52 (1: 4 Acetone-Hexane); [α]_D²⁵-46.8 ° (c0.37, CHCl₃); IR (neat): 2959,2932,2876,1734,1697,1460,1397,1366,1341,1283,1258,1167,1136,1098 and 772cm^-1;¹H-NMR (CDCl₃, 300MHz): 3.5-4.1 (m, 4H, NH, CH-OMe, OCH₂), 3.40 (s, 3H, OMe), 3.2 (m, 2H, N-CH₂), 2.45 (m, 1H, CH-CO), 1.55-2.0 (m, 4H, 2 × dap CH₂), 1.46, 1.56 (s, 9H, t-Bu), 1.32 (m, 6H, 3 × CH₂), 1.21 (d, J = 6.8Hz, 3H, CH₃) And 0.88 (t, J = 6.9H, 3H, CH₂-CH₃); EIMS (m / z): 325 (M⁺-MeOH, 4), 284 (1), 225 (1), 171 (3), 170 (27), 169 (2), 168 (1), 158 (1), 154 (1), 138 (5) , 136 (1), 126 (1), 118 (1), 117 (10), 115 (7), 114 (95), 113 (1), 110 (4), 103 (2), 86 (2) , 85 (4), 83 (1), 82 (3), 70 (100) and 57 (66%).

【0041】

T-Boc-Draploin (6) and octyliodide (7b) are reacted according to General Method C to synthesize t-Boc-Dap octyl ester (8b), and the obtained residue is eluted with 1: 3 acetone-hexane. Purify on a silica gel column to give the desired octyl ester as a colorless liquid (8b, 63%). Rf0.56 (1: 4 Acetone-Hexane); [α]_D²⁵-39.5 ° (c0.76, CHCl₃); IR (neat): 2957,2930,2874,2859,1734,1698,1458,1395,1366,1341,1256,1167,1136,1099 and 772cm^-1;¹H-NMR (CDCl₃, 300MHz): 3.5-4.1 (m, 4H, N-CH, CH-OMe, OCH₂), 3.40 (s, 3H, OMe), 3.21 (m, 2H, N-CH₂), 2.45 (m, 1H, CH-CO), 1.55-2.0 (m, 4H, 2 × dap CH₂), 1.46,1.60 (s, 9H, t-Bu), 1.24 (m, 15H, 6 × CH₂CH-CH₃) And 0.85 (t, J = 6.9Hz, 3H, CH₂-CH₃); EIMS (m / z): 367 (M⁺-MeOH, 4), 326 (2), 298 (1), 267 (2), 170 (33), 169 (2), 158 (2), 154 (2), 138 (5), 136 (1) , 126 (2), 118 (1), 117 (8), 116 (10), 115 (8), 114 (100), 113 (2), 103 (2), 86 (2), 85 (4) , 83 (2), 82 (3), 70 (78) and 57 (56%).

【0042】

T-Boc-Draploin (6) and hexylamine (7c) are reacted according to General Method D to form t-Boc-Dap-hexylamine (8c), and the obtained residue is used as an eluent using 1: 4 acetone-hexane. Purification on a silica gel column gives the desired hexylamide as a colorless liquid (8c; 90%). R0.25 (1: 4 Acetone-Hexane); [α]_D²⁵-47.1 ° (c0.21, CHCl₃); IR (neat): 3308,2965,2932,2874,1695,1670,1649,1549,1456,1400,1366,1286,1256,1227,1171,1105,1063,668,773 and 775cm^-1;¹H-NMR (CDCl₃, 300MHz): 6.26, 5.65 (brs, 1H, NH), 3.3-3.9 (m, 2H, N-CH, CH-OMe), 3.41 (s, 3H, OMe), 3.20 (m, 4H, 2 × N) -CH₂), 2.35 (m, 1H, CH-CO), 1.55-2.0 (m, 4H, 2 × dap CH)₂), 1.46,1.61 (s, 9H, t-Bu), 1.26 (m, 11H, 4 × CH, CH-CH₃) And 0.85 (t, J = 7.0Hz, 3H, CH₂-CH₃); EIMS (m / z): 338 (M⁺-MeOH); 297,269,238,210,201,186,170,154,138,114,111,91,70 (100%) and 57.

【0043】

General method E The synthesis of tripeptide trifluoroacetate salt (9) is carried out as follows. Trifluoroacetic acid (2 mL) is added to a solution of trypeptide t-putyl ester (5; 0.1 mM) in dichloromethane (2 mL) cooled to ice bath temperature in an argon atmosphere, and the solution is stirred at the same temperature for 1 hour. The solvent is then removed under reduced pressure to dissolve the residue in toluene and the solvent is removed again under reduced pressure. The residue is dried under vacuum to give the tripeptide trifluoroacetate salt (10) as a pale yellow sticky mass.

【0044】

General method The synthesis of FDAP ester / amide trifluoroacetate salt (10a-c) is carried out as follows. Trifluoroacetic acid (2 mL) was added to a t-Boc-Dap ester / amide (8a-c; 0.1 mM) solution in dichloromethane (2 mL) cooled to ice bath temperature under an argon atmosphere, and the solution was kept at the same temperature for 1 hour. Stir. The solvent is removed under reduced pressure, the residue is dissolved in toluene and the solvent is removed again under reduced pressure. The residue is dried under vacuum to give a pale yellow sticky mass of the corresponding Dap ester / amide trifluoroacetate salt (10a-c).

【0045】

General method The synthesis of G tetrapeptide ester / amide (12,14,16a-c) is carried out as follows. Dipeptide or Dap-ester / amide tfa salt (11,13,10a-c; 0.1 mM) and trypeptide tfa salt (9,,) in dry dichloromethane (2 mL) cooled to ice bath temperature (0-5 ° C) 15; 0.1 mM) Add triethylamine (4 mM) and then diethylcyanophosphate (1.1 mM) to the solution. Stir the solution at the same temperature for 1-2 hours. The solvent is removed under reduced pressure and the residue is chromatographed on a silica gel column with the solvent below to give the corresponding pentapeptide or tetrapeptide ester / amide (12, 14 and 16a-c).

【0046】

Compound 12 is synthesized as follows. Combined with dipeptide tfa salt (11) and trypeptide tfa salt (9) according to General Method G, purified on silica gel column with 3: 2 acetone-hexane as eluent and the corresponding pentapeptide (12; 55) in a colorless solid. %) Is obtained. Melting point 103 ~ 7 ° C; Rf0.55 (acetone-hexane 3: 2); [α]_D²⁵-67.5 ° (c0.08, CHCl₃); IR (thin film): 3295,2965,2934,2878,1620,2535,1499,1452,1418,1281,1202,1136 and 1099 cm^-1EIMS (m / z): 798 (M⁺, 2), 756 (2), 755 (4), 707 (1), 496 (1), 495 (5), 459 (1), 458 (2), 303 (1), 242 (1), 241 (8), 231 (1), 214 (1), 213 (3), 205 (1), 198 (1), 198 (1), 188 (10), 187 (1), 186 (10), 170 (4), 169 (2), 168 (2), 155 (1), 154 (6), 140 (2), 138 (3), 128 (4), 114 (1), 113 (3), 112 (2), 110 (1), 102 (26) and 101 (100%).

【0047】

Compound 14 is synthesized as follows. The dipeptide tfa salt (13) and the tripeptide tfa salt (9) are combined according to General Method G and purified on a silica gel column using 3: 2 acetone-hexane as an eluent to obtain the desired pentapeptide and a colorless concentrated liquid. (14; 86%). Rf0.55 (acetone-hexane 3: 2); [α]_D²⁵-45 ° C (c0.06, CHCl₃); IR (thin film): 3314,3300,2967,2934,1744,1640,1628,1545,1441,1414,1381,1277,1202,1167,1098,1038 and 984 cm^-1EIMS (m / z): 757 (M⁺, 1), 715 (2), 714 (6), 496 (1), 495 (5), 417 (1), 214 (4), 213 (1), 186 (4), 170 (2), 154 (2), 138 (1), 128 (2), 127 (2), 102 (10) and 101 (100%).

【0048】

Dov-val-Dil-Dap pentyl ester (16a) is synthesized as follows. Dap pentyl ester tfa salt (10a) is combined with tripe tfa salt (15) according to General Method G and chromatographed on a silica gel column using 3: 2 hexane-acetone as an eluent to make the desired tetrapeptide ester colorless. Obtained as a thick liquid (16a; 30%). Rf0.39 (Hexane-Acetone 3: 2); [α]_D²⁵-69.1 ° (c0.23, CHCl₃); IR (thin film): 3312,3295,2961,2934,2876,1728,1640,1452,1412,1389,1262,1200,1169,1132,1098 and 1038 cm^-1EIMS (m / z): 668 (M⁺, 1), 625 (2), 482 (3), 227 (3), 154 (2), 128 (2), 102 (9) and 101 (100%).

【0049】

Dov-val-Dil-Dap octyl ester (16b) is synthesized as follows. Dap pentyl ester tfa salt (10b) is combined with trypeptide tfa salt (15) according to General Method G and purified on a silica gel column using 1: 1 hexane-acetone as an eluent to make the desired tetrapeptide ester colorless. Obtained as a thick liquid (16b, 99%). Rf0.23 (Hexane-Acetone 3: 1); [α]_D²⁵-51.3 ° (c0.08, CHCl₃); IR (thin film): 3295,2961,2932,2876,2834,1730,1643,1622,1526,1454,1416,1385,1343,1304,1262,1200,1173,1134,1099,1038 and 721cm^-1EIMS (m / z): 710 (M⁺, 0.7), 667 (2), 481 (3), 227 (4), 199 (1), 186 (4), 184 (0.9), 155 (1), 154 (2), 128 (2), 117 (1), 102 (10) and 101 (100%).

【0050】

The compound Dov-val-Dil-Dap hexylamide (16c) is synthesized as follows. Dap-hexylamide tfa salt (10c) is combined with tripeptide tfa salt (15) according to General Method G and chromatographed on a silica gel column using 3: 2 hexane-acetone as an eluent to obtain the desired tetrapeptide amide. Obtained as a colorless, concentrated liquid (16c, 65%). Rf0.23 (3: 2 hexane-acetone); [α]_D²⁵-48.8 ° (c0.5, CHCl₃); IR (thin film): 3308,3295,2961,2930,2876,1620,1545,1452,1416,1383,1200,1167,1134 and 1099 cm^-1EIMS (m / z): 681 (M⁺), 666,650,638,525,481,449,412,355,341,269,253,227,214,199,186,170,154,128,114,102 and 101 (100%).

【0051】

To aid the understanding of the present invention, but not in a limited sense, the following examples are given.

【0052】

Example III-at-Boc-Dap Butyl Ester (8a) According to General Method C, t-Boc-drabroin (6) is reacted with pentyliodide (7a), and 1: 3 acetone-hexane is used as an eluent in a silica gel column. Purify above to give the desired octyl ester as a colorless liquid (8a, 50%); Rf0.52 (1: 4 acetone-hexane); [α]_D²⁵-46.8 ° (c0.37, CHCl₃); IR (neat): 2959,2932,2876,1734,1697,1460,1397,1366,1341,1283,1258,1167,136,1098 and 772cm^-1;¹H-NMR (CDCl₃, 300MHz): 3.5-4.1 (m, 4H, N-CH, CH-OMe, OCH₂), 3.40 (s, 3H, OMe), 3.2 (m, 2H, N-CH₂), 2.45 (m, 1H, CH-CO), 1.55-2.0 (m, 4H, 2 × dap CH₂), 1.46, 1.56 (s, 9H, t-Bu), 1.32 (m, 6H, 3 × CH₂), 1.21 (d, J = 6.8Hz, 3H, Me) and 0.88 (t, J = 6.9Hz, 3H, CH₂-CH₃); EIMS (m / z): 325 (M⁺-MeOH, 4), 284 (1), 225 (1), 171 (3), 170 (27), 169 (2), 168 (1), 158 (1), 154 (1), 138 (5) , 136 (1), 126 (1), 118 (1), 117 (10), 115 (7), 114 (95), 113 (1), 110 (4), 103 (2), 86 (2) , 85 (4), 83 (1), 82 (3), 70 (100) and 57 (66%).

【0053】

Example III-bt-Boc-Dap Octyl Ester (8b) React t-Boc-Drabroin (6) with Octyliodide (7b) according to General Method C, and elute the obtained residue with 1: 3 acetone-hexane. The desired octyl ester is obtained as a colorless liquid (8b, 63%); Rf0.56 (1: 4 acetone-hexane); [α]._D²⁵-39.5 ° (c0.76, CHCl₃); IR (neat): 2957,2930,2874,2859,1734,1698,1458,1395,1366,1341,1256,1167,1136,1099 and 772cm^-1;¹H-NMR (CDCl₃, 300MHz): 3.5-4.1 (m, 4H, N-CH, CH-OMe, OCH₂), 3.40 (s, 3H, OMe), 3.21 (m, 2H, N-CH₂), 2.456 (m, 1H, CH-CO), 1.55-2.0 (m, 4H, 2 × dap CH)₂), 1.46,1.60 (s, 9H, t-Bu), 1.24 (m, 15H, 6 × CH₂, CH-CH₃) And 0.85 (t, J = 6.9Hz, 3H, CH₂-CH₃); EIMS (m / z): 367 (M⁺-MeOH, 4), 326 (2), 298 (1), 267 (2), 170 (33), 169 (2), 158 (2), 154 (2), 138 (5), 136 (1) , 126 (2), 118 (1), 117 (8), 116 (10), 115 (8), 114 (100), 113 (2), 103 (2), 86 (2), 85 (4) , 83 (2), 82 (3), 70 (78) and 57 (56%).

【0054】

Example III-ct-Boc-Dap-hexylamide (8c) React t-Boc-drabroin (6) with hexylamine (7c) according to General Method D, and elute the obtained residue with 1: 4 acetone-hexane. The desired hexylamide is produced as a colorless liquid on a silica gel column (8c, 80%). Rf0.25 (1: 4 Acetone-Hexane); [α]_D²⁵-47.1 ° (c0.21, CHCl₃); IR (neat): 3308,2965,2932,2874,1695,1670,1649,1549,1456,1400,1366,1286,1256,1227,1171,1105,1063,668,773 and 725cm^-1;¹H-NMR (CDCl₃, 300MHz): 6.26, 5.65 (brs, 1H, NH), 3.3-3.9 (m, 2H, N-CH-CH-OMe), 3.41 (s, 3H, OMe), 3.20 (m, 4H, 2 × N) -CH₂), 2.35 (m, 1, CH-CO), 155-2.0 (m, 4H, 2 × dap CH₂), 1.46,1.61 (s, 9H, t-Bu), 1.26 (m, 11H, 4 × CH₂, CH-CH₃) And 0.85 (t, J = 7.0Hz, 3H, CH₂-CH₃); EIMS (m / z): 338 (M⁺-MeOH), 297,269,238,210,201,186,170,154,128,114,111,91,70 (100%) and 57.

【0055】

Example VIDov-Ile-Dil-Dap-Doe (12) Silica gel column with dipeptide tfa salt (11) and tripeptide tfa salt (9) bonded according to General Method G and acetone-hexane (3: 2) as eluent. The desired pentapeptide produced above is obtained as a colorless solid (12; 55%). Melting point 103 ~ 107 ° C; Rf0.55 (acetone-hexane 3: 2); [α]_D²⁵-67.5 ° (c0.08, CHCl₃); IR (thin film): 3295,2965,2934,2878,1620,1535,1499,1542,1381,1202,1136 and 1099 cm^-1EIMS (m / z): 798 (M⁺, 2), 756 (2), 755 (4), 707 (1), 496 (1), 695 (5), 459 (1), 458 (2), 303 (1), 242 (1), 241 (8), 231 (1), 214 (1), 213 (3), 205 (1), 198 (1), 188 (10), 187 (1), 186 (10), 170 (4), 169 (2), 168 (2), 155 (1), 154 (6), 140 (2), 138 (3), 128 (4), 114 (1), 113 (3), 112 (2), 110 (1), 102 (26) and 101 (100%).

【0056】

Example VIIDov-Ile-Dil-Dap-Met-OMe (14) Combine dipeptide tfa salt (13) and trypeptide tfa salt (9) according to General Method G, and use acetone-hexane (3: 2) as an eluent. Purification on a silica gel column to give the desired pentapeptide as a thick, colorless liquid (14; 86%); Rf0.55 (acetone-hexane 3: 2); [α]_D²⁵-45 ° (c0.06, CHCl₃); IR (thin film): 3314,3300,1967,2934,1744,1644,1640,1628,1545,1441,1414,1381,1277,1202,1167,1098,1038 and 984 cm^-1EIMS (m / z): 757 (M⁺, 1), 715 (2), 714 (6), 496 (1), 495 (5), 417 (1), 241 (4), 213 (1), 186 (4), 170 (2), 154 (2), 138 (1), 128 (2), 127 (2), 102 (10) and 101 (100%).

【0057】

Example VIII-a Dov-val-Dil-Dap pentyl ester (16a) Dap pentyl ester (10a) and trypeptide tfa salt (15) are combined according to General Method G, and hexane-acetone (3: 2) is used as an eluent. Chromatography on a silica gel column to give the desired tetrapeptide ester as a thick, colorless liquid (16; 30%); Rf0.39 (hexane-acetone 3: 2); [α]_D²⁵-69.1 ° (c0.23, CHCl₃); IR (thin film): 3312,3295,2961,2934,2876,1728,1640,1452,1412,1389,1262,1200,1169,1132,1098 and 1038 cm^-1EIMS (m / z): 668 (M⁺, 1), 625 (2), 482 (3), 227 (3), 154 (2), 128 (2), 102 (9) and 101 (100%).

【0058】

Example VIII-b Dov-val-Dil-Dap Octyl Ester (16b) Combines Dap pentyl ester tfa salt (10b) and trypeptide tfa salt (15) according to General Method G and elutes hexane-acetone (1: 1). Used as a liquid and chromatographed on a silica gel column to give the desired tetrapeptide ester as a dark colorless liquid (16b, 99%); Rf0.23 (hexane-acetone 3: 1); [α]_D²⁵-51.3 ° (c0.08, CHCl₃); IR (thin film): 3295,2961,2932,2876,2834,1730,1643,1622,1526,1454,1416,1385,1343,1204,1262,1200,1173,1134,1099,1038 and 721cm^-1EIMS (m / z): 710 (M⁺, 0.7), 667 (2), 481 (3), 227 (4), 199 (1), 186 (4), 184 (0.9), 155 (1), 154 (2), 128 (2), 117 (1), 102 (10) and 101 (100%).

【0059】

Example VIII-c Dov-val-Dil-Dap Hexylamide (16c) Combine Dap-hexylamide tfa salt (10c) and trypeptide tfa salt (15) according to General Method G to add hexane-acetone (3: 2). Chromatographically treated on a silica gel column as an eluent to give the desired tetrapeptide amide as a dark colorless liquid (16c, 65%); Rf0.23 (hexane-acetone 3: 2); [α]_D²⁵-48.8 ° (c0.5, CHCl₃); IR (thin film): 3308,3295,2961,2930,2876,1620,1545,1535,1452,1416,1383,1200,1167,1134 and 1099 cm^-1EIMS (m / z): 681 (M⁺), 666,650,638,525,481,449,412,355,341,269,253,227,214,199,186,170,154,128,114,102 and 101 (100%).

【0060】

From the above, it is clear that the useful embodiments of the present invention have been described and described to satisfy all of the aforementioned objects of the significantly unpredictable aspects. It should be appreciated that modifications, changes and changes that are readily encountered by those skilled in the art in this disclosure are within the spirit of the statement, which is limited solely by the claims.