In!. J. Peptide Protein Res. 41, 1993, 282-290
`
`Serine-containing 10-membered cyclodepsipeptides
`
`Synthesis and molecular structure of PhCH2CO-DSer-Pro-Pro; and
`I
`PhCHlCO-DSer-Pro-DPro-
`
`S. CERRINI I. E. GAVUZZO ', G. LUIS1 and F. PINNEN3*
`G . Giaconiello Insrirute of Srrucrural Chemisrrj,. C. N . R . , Monrerorotido Stazione, Rome, Italy and Departrnertt qf
`Phartnuceuticul Srudies. La Sapienza University, Rorne, Ira!).. Instirirre of Pharmaceutical Chemistry, University of Catania,
`Caratria. Ira!\*
`
`Received 31 May, accepted for publication 31 July 1992
`
`- -
`
`As a part of a research program aimed at studying synthesis and conformation of small ring peptides, the
`cyclization of diastereoisomeric N-phenylacetyl-seryl-propyl-proline tripeptides has been examined. Two 10-
`membered peptide lactones, PhCHzCO-DSer-Pro-Pro- 5a and PhCH2CO-DSer-Pro-DPro- 5b, have been
`isolated by treating the corresponding linear p-nitrophenyl esters with DBU in dry benzene. In these two
`compounds the serine lactone fragment (a common structural feature of several bioactive cyclodepsipeptides)
`is inserted into a highly strained small ring system. The conformation in the crystal of 5a and 5b has been
`studied by X-ray analysis. Both the 10-membered rings of 5a and 5b adopt an overall cis-cis-trans confor-
`mation in which the lactone junction is rrans. The deviations from planarity of the peptide units vary from
`for the DSer-PrO bond in 5a and PrO-DPrO bond
`d w = 30" for the DSer-Pro bond in 5b to Ao= 5-6'
`in 5b. The skeletal atoms of 5b, containing the PrO-DPrO sequence, are related by a pseudo-symmetry mir-
`ror plane passing through the Pro carbonyl and the opposite DSer C
`
` Zgroup. In both the molecules the
`
`~
`exocyclic amide bond adopts an extended conformation with respect to the DSer-Pro ring junction; this ar-
`rangement gives rise to a Cs-type ring structure which is well evidenced in the case of 5a.
`
`Key words: conformation; cyclodepsipeptides: peptide lactones; proline; serine; tripeptides; X-ray structure
`
`The present report is a part of our continuing interest
`in the chemistry and conformation of cyclotripeptides
`and cyclotridepsipeptides (tripeptide lactones) (1 -6).
`These compounds represent, after dioxopiperazines
`and dioxomorpholines, the smallest cyclopeptide sys-
`tems and are characterized by unusual chemical and
`structural properties, basically connected with the con-
`
`Abbreviations follow the recommendations of the IUPAC-ICB Com-
`mission on Biochemical Nomenclature as given in Eur. J . Biochem.
`(1984) 138, 9-37. Additional abbreviations: AcOH. acetic acid;
`DBU, 1.8-diazabicyclo[ 5,4,0]undec-7-ene; DCCI, dicyclohcxylcarbo-
`diimide: DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; HOBt,
`I-hydroxybenzotriazole; Hyb, S-3-hydroxybutyric acid; MeAnt.
`N-methylanthranilic acid; MeOH, methanol; NNM, N-methyl-
`morpholine; THF, tetrahydrofuran; TLC, thin-layer chromatogra-
`phy; TMS, tetramethyl silane.
`
`282
`
`formational constraint of the nine-membered ring, de-
`viation from planarity of the peptide bonds and short
`transannular distances leading to isomerizations and
`rearrangements. In this field we described previously
`synthesis and conformational preferences of a group of
`10-membered cyclotripeptides and cyclotridepsipep-
`
`-
`
`tides possessing the general structure -/?Xaa-Yaa-Pro-,
`where BXaa represents a p-amino- or a 8-hydroxy-acid
`residue (1, 2,4-6). These systems are characterized by
`low conformational heterogeneity, and present, as com-
`pared with the well known nine-membered cyclotrip-
`eptides, a less pronounced propensity towards transan-
`nular interactions and molecular rearrangements. This
`feature allowed the design of valuable models for de-
`tailed conformational investigations (6).
`As a continuation of these studies we have examined
`
`IPR2014-01126- Exhibit 1031 p. 1
`
`

`
`the synthesis and conformation of ten-membered trip-
`I - ?
`eptide lactones of the R-CO-Ser-Pro-Pro- type, con-
`taining two proline residues in addition to a serine unit
`as the P-hydroxyacidic component. As previously
`pointed out by Mauger & Stuart (7), the lactone unit in
`which a serine or threonine side-chain is 0-acylated by
`the carboxy terminus of the peptide is a key structure
`feature of several bioactive natural compounds. In these
`peptide lactones the exocyclic serine or threonine amino
`group provides the handle to which a carboxylic acid,
`usually responsible for the activiti, is bound. This is the
`case of quinoxaline antibiotics (8) and siderophore en-
`terobactin, a trimer lactone of N-2,3-dihydroxybenzoyl
`serine (9). In more strained and complex systems, such
`as the tyrosinase inhibitor microviridin (lo), the serine
`residue is part of a peptide lactonc, bridged through the
`serine amino group to a larger tricyclic peptide struc-
`ture.
`7 7
`In the case of the RCO-Ser-Pro-Pro- models reported
`here, the general lactone fragment has been inserted
`into a highly strained ring system in order to reduce the
`conformational variability of the peptide backbone and
`gain information on the spatial relationship between the
`backbone elements and the exocyclic carboxyamide
`moiety. To our knowledge these compounds represent
`the smallest cyclodepsipeptide models obtained through
`the “side chain to end” mode of cyclization (1 1).
`
`RESULTS AND DISCUSSION
`At variance with cyclopeptides containing at least one
`secondary amide bond in the ring, the present models
`cannot be synthesized by using the hydroxyacyl inser-
`tion method, a strategy which minimizes the oligomer-
`ization reactions frequently encountered during the syn-
`thesis of small ring cyclopeptides. Thus, the end-to-end
`cyclization through ester bond formation between the
`free serine 8-hydroxyl and the activated carboxylic
`group of the C-terminal proline was examined.
`In a first approach the homochiral N-phenylacetyl
`tripeptide PhCHlCO-Ser-Pro-Pro-ONp was selected
`as suitable linear precursor. Adoption of different cy-
`clization conditions led, however, to unsatisfactory re-
`sults owing to the formation of cyclo-oligomers, among
`which the 40-membered tetrameric peptide lactone was
`the prevailing species (12). The two heterochiral iso-
`mers Ph-CH2-C0-~Ser-Pro-Pro-ONp and Ph-CH2-
`CO-DSer-Pro-DPro-ONp, 4a and 4b, respectively, cor-
`responding to the DLL and DLD sequences, were then
`synthesized and subjected to cyclization (Fig. 1). In
`contrast to the results obtained with the homochiral
`precursor, both 4a and 4b gavc, by treatment with
`
`DBU in dry benzene (3), good yields of the correspond- -
`
`lactones PhC112CO-~Ser-Pro-Pro-
`ing monomeric
`m
`5a and PhCH2CO-DSer-Pro-DPro- 5b. Note that,
`since the serine side-chain is involved in the ring sys-
`
`Serine-containing 10-membered cyclodepsipeptides
`
`li
`
`1a.b
`
`5a,b
`
`FIGURE 1
`Synthesis of cyclotridepsipeptides 5a and 5b. Compounds a and b
`contain the sequence Pro-Pro and PrO-DPrO, rcspcctivcly. Reagents:
`(i) DCCI, HOBt; (ii) 2 N NaOH; (iii) Hz, 5 % Pd/C; (iv) DCCI,
`p-nitrophenol; (v) DBU.
`
`tem instead of the serine a-amino group, the PhCHrCO-
`m
`DSer-Pro-Pro- heterochiral stereoisomer 5a presents
`all three a-hydrogen atoms on the same side of the
`mean plane of the cyclopeptide ring, a stereochemical
`feature typical of homochiral cyclopeptides. Formation
`of
`the
`isomeric 0-phenylacetyl homodetic nine-
`membered cyclotripeptide resulting from an N to 0 acyl
`shift, a frequently encountered side-reaction at the Ser
`and Thr residues (13), is not observed. In accordance
`with the assigned lactone structure, treatment of 5a,b
`with hydrazine hydrate gives only the corresponding
`N-phenylacetyl tripeptide hydrazides; no traces of
`PhCH2CO-NHNH2 are evidenced.
`Crystals of compounds 5a and 5b suitable for crys-
`tal structure determination were grown from EtOAc
`solutions by slow evaporation. A perspective view of
`the molecular structures of 5a and 5b is reported in
`Figs. 2 and 3; the fractional atomic coordinates of both
`compounds are reported in Table 1. Bond lengths and
`bond angles (Table 2) are in general agreement with the
`previously reported values for proline-containing small
`ring cyclopeptides (1-6, 14). Attention should be drawn
`to the high value of the ring angles at NZ in 5a and N3
`in 5b, both corresponding to cis-planar peptide nitrogen
`atoms. Deviations from the standard values are also
`shown by the ring angles at the tetrahedral carbon atom
`C; in 5b (Q), at the sp’ carbon atoms C’Z and C’j in
`5b and at C’3 in 5a. A significant compression of the
`tetrahedral angle is observed in 5a at the extra-ring
`angle of DSer C;* atom (71; Table 2). Interatomic dis-
`tances across the ring which are significantly shorter
`
`283
`
`IPR2014-01126- Exhibit 1031 p. 2
`
`

`
`Cerrini et al.
`
`O H oc
`
`O N
`
`0 0
`
`O H
`o c
`0"
`
`0 0
`
`FIGURE 2
`
`FIGURE 3
`r
`r
`A perspective view of the molecular structure of Ph-CH2-CO-DScr-
`A perspective view of the molecular structure of Ph-CH2-CO-DSer-
`I
`1
`Pro-DPro- 5b. The outlined bonds concern disordered proline C;
`Pro-Pro- 5a. The outlined bonds concern disordered proline C ;
`atoms.
`atoms.
`
`than the sum of the van der Waals radii involve, in both
`the molecules, the atoms of the lactone unit (0: in 5a
`and C; in 5b); a short contact is also observed in 5b
`between the two a-hydrogen atoms of the fragment
`DSer-Pro Figs. 3 and 4 and Table 3).
`The backbone conformation of 5a and 5b is described
`by the sequence of torsion angles reported in Table 4.
`The conformation of the exocyclic amide bond is trans
`in both 5a and 5b, with a significant distortion from
`planarity in the case of 5a (04 = 168.0'). Each ring is
`characterized by the presence of two cis-configurated
`peptide bonds (01
`and 02) and by a trans lactone unit
`( ~ 3 ) . Thus, the overall backbone conformation of the
`two rings can be described as cis-cis-trans. An analo-
`gous arrangement of the three CO-X (X = N; 0) ring
`junctions has been found in all the ten-membered ho-
`modetic and heterodetic cyclotripeptides studied so far,
`with the notable exception of cyclo(-MeAnt-Phe-Pro-)
`(4), which adopts an all-cis backbone conformation
`analogous to the symmetrical crown conformation
`adopted by the homochiral nine-membered cyclotrip-
`eptides (14).
`The conformational constraint of ten-membered ring
`induces different distortion on the three ring junctions.
`Thus, in both 5a and 5b (Table 4) one of the two amide
`bonds is practically planar ( d w z So), whereas the other
`
`284
`
`4
`
`FIGURE 4
`
`Side view of the cyclic backbone of Ph-CH2-CO-DSer-Pro-DPrd 5b,
`showing the pseudo-symmetry mirror plane. Backbone bonds are
`printed in bold-face.
`
`deviates significantly from planarity; in particular, the
`DSer-Pro bond of 5b (do, = 30.1 ") presents one of the
`largest deviations observed so far. The CO-0 lactone
`group adopts, in both models, a significantly distorted
`trans-conformation [ do3 = 2 1.4 (5a) and 25.1 O (5b)],
`
`IPR2014-01126- Exhibit 1031 p. 3
`
`

`
`Atom
`
`Serine-containing 10-membered cyclodepsipeptides
`
`TABLE 1
`Fractional tiloniic coordinates (with e.s.d.s in parentheses) and thermal parameters
`
`X
`
`0.7194(6)
`0.605( 1)
`0.589( 1)
`0.479( 1)
`0.444( 1)
`0.2463(7)
`0.5333(7)
`0.7565( 9)
`0.805( 1)
`0.577( 1)
`0.402( 1)
`0.738( 1)
`0.5886(7)
`0.8973(9)
`I .054( 1)
`1.184(1)
`1.026(3)
`1.1 13(3)
`0.886( I )
`0.942( 1)
`1.0472(8)
`0.584( 1)
`0.784( 1)
`0.445(2)
`0.413(1)
`0.585( 1)
`0.210( 1)
`0.554( 1)
`0.178(2)
`0.347(2)
`
`0.2979(5)
`0.2275(7)
`0.3407(6)
`0.2750(6)
`0.4625(6)
`0.45 14(6)
`0.5734(5)
`0.6302(7)
`0.7955(8)
`0.8 173(9)
`0.6875(7)
`0.5938(6)
`0.6288(6)
`0.5343(6)
`0.4804(8)
`0.430( 1)
`0.386(2)
`0.464(3)
`0.503( 1)
`0.3592(8)
`0.3226(7)
`0.1803(7)
`0.1360(6)
`
`1'
`
`0.1623(3)
`0.1026(5)
`- 0.0151(4)
`- 0.0788(4)
`- 0.0302(4)
`- 0.0569(3)
`- 0.0169(3)
`0.0205(4)
`- 0.051 l(5)
`- 0.0729(5)
`- 0.0520(5)
`0.1402(4)
`0.1665(3)
`0.2107(3)
`0.1979(4)
`0.3054(6)
`0.384( 1)
`0.366( 1)
`0.3247(4)
`0.1735(4)
`0.1617(4)
`- 0.1280(5)
`-0.1144(5)
`- 0.2073(5)
`- 0.1767(4)
`- 0.1899(4)
`- 0.1410(7)
`- 0.1691(5)
`- 0.1220(8)
`- 0.1361(7)
`
`0.7260
`0.6849(9)
`0.6867(8)
`0.6487(7)
`0.5985(7)
`0.4930(6)
`0.6389(7)
`0.7630(7)
`0.7444(9)
`0.625( 1)
`0.5493(9)
`0.8468(7)
`0.9524(6)
`0.8 104(7)
`0.6919(7)
`0.714(1)
`0.840( 1)
`0.827(2)
`0.9022(9)
`0.6423 (8)
`0.5372(7)
`0.7 196(8)
`0.8160(7)
`
`0.4303( 1)
`0.4693(2)
`0.4523(2)
`0.4906(2)
`0.4062(2)
`0.4 109( 1)
`0.3610( 1)
`0.3458(2)
`0.3007(2)
`0.2775(2)
`0.3 177(2)
`0.3291(2)
`0.3009( I )
`0.344 l(2)
`0.3854(2)
`0.3869(3)
`0.3674(5)
`0.3396(6)
`0.3271(2)
`0.4348(2)
`0.47 15( 1)
`0.5274(2)
`0.5346(2)
`0.5565(2)
`0.6101(2)
`0.6438(2)
`0.6266(3)
`0.6932(2)
`0.6772(4)
`0.7092(3)
`
`0.2379(4)
`0.3 358(6)
`0.4453(5)
`0.5463(5)
`0.4258(5)
`0.4522(4)
`0.3 694(4)
`0.3701(5)
`0.39 19(7)
`0.3295(9)
`0.3520(6)
`0.2632(5)
`0.2793(5)
`0.1559(4)
`0.1147(5)
`- 0.0148(6)
`- 0.027( 1)
`- 0.048(2)
`0.0632(6)
`0.1764(6)
`0.1682(5)
`0.5926(6)
`0.5483(5)
`
`B(eq).'
`
`3.9(1)
`4.3(1)
`3.9(1)
`5.2(1)
`3.7(1)
`4.6(1)
`3.4(1)
`3.3(1)
`4.1(1)
`4.8(2)
`4.4( 1)
`3.3(1)
`4.6(1)
`4.0( I )
`4.0( 1)
`6.8(2)
`5.9( 4)h
`5.3(5)b
`5 3 2 )
`3.8(1)
`5.6(1)
`4.8(2)
`6.9(2)
`6.0(2)
`4.1(1)
`4.0(1)
`7.0(2)
`5.3(2)
`9.2(3)
`6.9(2)
`
`4.2(1)
`4.4(2)
`3.6(1)
`3.8(1)
`3.3(1)
`4.4(1)
`3.2(1)
`3.4(1)
`5.2(2)
`6.3(3)
`4.3(2)
`3.5(1)
`4.8(1)
`3.9(1)
`4.0(2)
`7.0(3)
`4.6(4)
`5.1(6)
`5.5(2)
`4.2(2)
`6.1(2)
`4.1(2)
`5.3(1)
`
`285
`
`IPR2014-01126- Exhibit 1031 p. 4
`
`

`
`Cerrini et al.
`
`Atom
`
`X
`
`0.13 M(7)
`- 0.03 14(7)
`-0.105(1)
`- 0.1134(8)
`- 0.256( 1)
`- 0.2667(9)
`- 0.3357(8)
`0.945(3)
`0.970(3)
`0.997(2)
`0.811(3)
`
`a B(eq) = (4/3) C, u, . u, h,.
`' This atom was refined isotrop~cal'y.
`' Symbol A denotes solvent atoms (see text).
`
`TABLE 1
`iconrimred)
`
`1'
`
`0.6749(9)
`0.6676(8)
`0 5586(9)
`0.7692(8)
`0.552( 1)
`0.760( 1)
`0.653( 1)
`0 641(3)
`0.777(3)
`0.525(2)
`0 585(3)
`
`~
`
`0.7082(6)
`0.7025(5)
`0.6792(8)
`0.7 196(7)
`0.6762(8)
`0.7153(8)
`0.6940(7)
`0.0 16( 2)
`0.0 19(2)
`0.003(2)
`-
`0.028(2)
`
`_______
`
`B(eq)a
`
`4.7(2)
`3.6(1)
`5.2(2)
`4 3 2 )
`5.8(2)
`5.5(2)
`5 4 2 )
`15.9(7)','
`14.9(7)'.'
`1 2.0(5)b,c
`17.9( 9)b,c
`
`and this can be related to the low rotational barrier of
`TABLE 3
`inlrariiolecrilur short distances (i) for PhCHrCO-D-
`esters as compared with amides.
`Selecred
`Together with the distortion of the peptide junctions i
`Ser-Pro-Pro- 5a and PhCHzCO-DSer-Pro-DPro- 5b
`(do), the pyramidality of the nitcogen atoms, as re-
`vealed by the displacement d(N) (A) from the plane of
`its three substituents, has been determined. All the
`amide nitrogen atoms are practically flat, except the o i . . . .o:
`nitrogen atoms involved in the two distorted amide 0 ; . . . . c;
`bonds (N3 in 5a and Nz in Sb), whicb are both signif- c; . . . . c;
`in 5a and Nz ' . . C;
`icantly pyramidoal: d(N3) = 0.113 A
`d(N2) = - 0.201 A in 5b. It should be mentioned in this H;.
`. . H;
`Hz. . . H7
`H . . . . O
`N . . . . O
`
`-
`
`5a
`
`2.89
`2.76
`
`2.30
`2.27
`2.20
`2.58
`
`5b
`
`2.95
`2.78
`2.08
`
`2.35
`2.69
`
`context that, in agreement with the high deviation from
`planarity (do1 = 30.1 ") and the significant nitrogen py-
`ramidalization, the CO-N2 bond in 5b exhibits the
`greatest C'- N length (1.356 A; Table 2) (6).
`Both 5a and 5b have the pyrrolidine ring of the Pro?
`residue in the envelope (C, symmetry) conformation
`which can be described as C,-C"-endo in 5a and C,-
`Cfi-exo in 5b (1 5). As frequently found in crystal struc-
`tures of proline-containing peptides, the Cy atoms of
`Pro3 residues in both 5a and 5b are disordered (16). By
`taking into account the two positions assigned to the
`C j atoms in the crystal (Figs. 2 and 3), all the resulting
`Pro3 pyrrolidine rings can be described as half-chair (C2
`symmetry) conformations.
`Although several medium-sized cyclodepsipeptides
`have been studied, only few data are at the present
`available on small ring models. It thus seemed inter-
`esting to correlate the backbone conformation of 5a,b
`with that of the previously studied ten-membered cy-
`clotridepsipeptide -Hyb-Phe-Pro- (6) (l), characterized
`by the presence of only one proline residue in the
`
`-
`
`-
`
`TABLE 2
`Selected hond lengths (A) and ring
` angle^ l ' ) for P h C H 2 C O - ~ -
`m
`Ser-Pro-Pro- 5a arid PhCHzC0-~Ser-Pro-DPro- 56. with e.s.d.s in
`parentheses
`
`5a
`
`1.344(7)
`1.347(7)
`1.33 l(7)
`1.323(8)
`
`113.3(4)
`120.7(5)
`130.6(4)
`108.6(4)
`118.6(4)
`127.3(4)
`114.6(5)
`11 1.8(4)
`I16.6(4)
`107.2(4)
`lOS.3(5)
`
`5b
`
`1.356(8)
`1.348(9)
`1.333(8)
`1.335(9)
`
`108.0(6)
`118.4(7)
`126.6(6)
`I2 1.0(6)
`125.0(7)
`130.5(6)
`114.6(6)
`113.0(7)
`I17.4(5)
`108 l(5)
`109 l(6)
`
`Bonds
`Ci-N2
`Ci-NX
`c;-0;
`Ci-Ni
`Angles
`c+c;-c;
`C;-C; -Nz
`C -Nz-C?
`N2-cS-ci
`C;-C;--Nl
`C;-NX-C;
`N,-C;-C;
`c;-c;-o'(
`c;-o;-cr:
`0i-Cf-C;
`Ni-CT-Ci
`
`286
`
`Tz
`
`~3
`
`Ti
`
`IPR2014-01126- Exhibit 1031 p. 5
`
`

`
`Selected
`
`Serine-containing 10-membered cyclodepsipeptides
`
`i
`
`W I
`c p ~
`
`$2
`
`~2
`
`( ~ 3
`
`$3
`
`Backbone
`0 {-Cf-Ci"-C
`Cf-C;-C;-N:
`CT-Ci-N,-CZ
`Ci-Nz-C;--Ci
`Nr-CI-Ci-N3
`C;-CI-N3-C;
`C&N3-C;-Ci
`N,-C;-C.i-O:
`c;-c;-o;-c.f W3
`C;-O;-Cf-C;
`Phenylacetyl side-chain
`c: -cp2;-c;
`C$--C;-C;-N
`C;-C;-Ni-CT
`Ci-Ni-CY-C;
`NI--CY-Ci-N2
`
`is perpendicular to the mean plane of the ring and
`TABLE 4
`passes through the Pro2 carbonyl group and the oppo-
`cycbitripeptides PhCH2CO-D-
`torsion angles (") of
`site DSer Cf atom. In the side view of Fig. 4, this sym-
`I r 7
`Ser-Pro-Pro- 5a. PhCH~cO-DSer- Pro-DPro- 56 and
`metrical ring conformation is clearly seen together with
`m
`the relative spatial orientation of the three u-hydrogen
`Hyb-Phe-Pro- 6
`atoms and the main plane containing the six atoms N2,
`Cq, C,, 02, N3, C;.
`In both molecules the exocyclic amide bond bearing
`the phenylacetyl group adopts an extended conforma-
`tion with respect to the DSer-Pro ring junction. An
`examination of the parameters involved in this portion
`of the molecule reveals short intraresidue NI . . . 0 1 dis-
`tances, N I H . * * 01C; hydrogen-bond contacts (Ta-
`ble 3) and high values of $1 and $1 torsion angles (Ta-
`ble 4). All these data indicate that the exocyclic serine
`amide N-H interacts with its endocyclic carbonyl group
`to give a Cs-type ring structure, analogous to that in-
`duced in peptide backbones by the presence of up-
`disubstituted glycine residues (17). In the case of 5a the
`Cs structure is supported by the characteristic com-
`pression of the tetrahedral angle at the C; atom in-
`volved in the pseudopentagonal ring (71 = 105.3'; Ta-
`ble 2) and by the observation that 5a is not involved in
`any intermolecular H-bonding scheme (17, 18). By tak-
`ing into account the reduction of conformational free-
`dom induced by the Cs ring structure, it can be argued
`that this factor may play a significant role in the great
`similarity of conformations that the external carboxya-
`mide groups adopt in the two models (Table 4).
`The crystal packing of 5a is mainly characterized by
`van der Waals interactions. In 5b, on the other hand,
`a weak intermolecular H bond relates the NIH hydro-
`gen atom with the carbonylic pxygen of a screw-related
`molecule (N1 *
`* . 0 2 = 3.01 A). This interaction gives
`rise to a packing characterized by a channel in which
`guest molecules of crystallization solvent can be located
`(see Fig. 5 and experimental section).
`
`5a
`
`- 64.1
`85.2
`- 5.9
`- 101.8
`136.4
`- 18.9
`- 54.2
`3.2
`158.6
`- 93.3
`
`- 75.8
`- 116.6
`- 168.0
`148.4
`- 155.8
`
`5b
`
`6
`
`- 69.9
`79.0
`6.6
`- 112.2
`136.9
`- 18.1
`- 43.6
`- 15.9
`163.6
`- 75.9
`
`- 62.1
`85.9
`30.1
`- 104.5
`- 13.2
`5.0
`59.3
`15.2
`- 154.9
`90.8
`
`- 82.5
`- 126.1
`- 173.2
`170.1
`- 154.2
`
`i
`
`WJ
`
`(PI
`
`$1
`
`ring. The sequence of the backbone torsion angles of 6
`reported in Table 4 shows a strikingly similarity of the
`conformation of this model with that adopted by 5a, the
`largest difference between the corresponding torsion
`angles being 19". The similarity between the backbone
`conformations of these two models, despite the pres-
`ence in 5a of two units of the conformationally con-
`strained residue of proline, suggests that the ring con-
`formation adopted by 5a and 6 may represent the
`preferred arrangement of ten-membered cyclotripep-
`tides containing two homochiral x-aminoacids.
`An examination of Fig. 3 and of the torsion angles
`reported in Table 4 shows that the skeletal atoms of 5b
`are related by a pseudo-symmetry mirror plane which
`
`*
`
`EXPERIMENTAL PROCEDURES
`
`Melting points are uncorrected. TLC was performed on
`precoated silica gel (Merck 60F 254 plates) developed
`
`FIGURE 5
`Stereoscopic view of the crystal packing of 5b.
`
`287
`
`IPR2014-01126- Exhibit 1031 p. 6
`
`

`
`Cerrini et al.
`
`with the following solvent systems: (A) Et0Ac:MeOH
`(95:5); (B) Et0Ac:AcOH (97:3), (C) CHC13:MeOH
`(97:3). Optical rotations were taken at 20°C with a
`Schmidt-Haensch Polartronic D polarimeter at the con-
`centration c = 1 in CHC13 (unless otherwise stated). IR
`spectra were recorded with a Perkin-Elmer 983 spec-
`trophotometer. 'H (300 MHz) and
`(75.43 MHz)
`NMR spectra were determined in CDC13 solution with
`TMS as internal standard using a Varian XL-300 in-
`strument.
`Fast atom bombardment (FAB) mass spectra of
`compounds 5a and 5b were recorded on a Kratos
`MS80RF mass spectrometer equipped with an Ion
`Tech B 11 saddle field gun operating at 8 kV and 36 pA
`of current using Xe to form energetic atoms.
`
`Prepurution of protected trQeptide rnethJ.1 esters l a arid
`l b (Fig. 1 )
`To a stirred solution of PhCHKO-DSer(Bz1)-OH
`(6.4 g, 20.5 mmol) in CH2C12 (80 mL), HOBt (5.5 g,
`41.0mmol) was added at room temperature. After
`10 min DCCl(4.2 g, 20.5 mmol) and after further 10 min
`the corresponding dipeptide methyl ester hydrochloride
`(Pro-Pro-OMe.HC1 for compound
`la; Pro-DPro-
`OMe.HCI for compound lb) (5.4 g, 20.5 mmol) and
`NMM (2.1 g, 20.5 mmol) in CH2C12 (55 mL) were
`added at 0°C. After 1 h at O'C and 2 h at room tem-
`perature the mixture was filtered and the resulting so-
`lution was washed with 2 N citric acid, saturated aque-
`ous Na2CO3 and HzO. The residue obtained after
`drying and evaporation was chromatographed on silica
`gel using Et0Ac:MeOH (95:5) as eluant to give methyl
`esters la and l b as oily products.
`
`l a . 8.0 g
`(75""
`PhCH2CO-DSer(Bzl)-Pro-Pro-OMe
`'H-NMR 6= 1.8-2.2
`yield). Rf(A)=0.6; [ r ] D -66.0'.
`(8H, m, 2 x Pro P-CH, and 2 x Pro ;'-CH2), 3.5 (2H, s,
`CH?CO), 3.5-3.7 (6H, m, 2 x Pro 6-CH2, Ser P-CH?),
`3.7 (3H, S, OCH3), 4.4 (2H, AB q, J = 12.4Hz,
`PhCH20), 4.5-4.65 (2H, m, 2 x Pro r-CH), 5.0 (1 H, m,
`Ser a-CH), 6.5 (lH, d, J = 8.5 Hz, NH), 7.1-7.3 ppm
`(10H, m, ArH). Anal. cak. for C29H35N306: C 66.78,
`H 6.76, N 8.06",. Found: C 66.85, H 6.80, N 8.03"".
`PhCHzCO-DSev(Bz~)-Pro-Pro-OMe l b . 6.4 g (60",). Rf
`(A) = 0.6; [ z ] ~ + 30.0'. 'H-NMR 6 = 1.8-2.4 (8H, ni,
`2 x Pro P-CH2 and 2 x P r o y-CHz), 3.55 (2H, s,
`CHXO), 3.4-3.8 (6H, m, 2 x Pro 6-CH2, Ser P-CHl),
`3.65 (3H, S , OCH3), 4.5 (2H, S, PhCHIO), 4.3-4.6 (2H,
`m, 2 x P r o r-CH),4.9(IH, m, Ser x-CH), 6.5 ( l H , d ,
`J = 7.5 Hz, NH), 7.35 ppm (10H, m, ArH). Anal. calc.
`for C29H35N306: C 66.78, H 6.76, N 8.06"". Found: C
`66.81, H 6.83, N 8.107,.
`
`Hydroijxis of 0-protecred tripeptide methJ.1 esters l a
`atid l b
`To a solution of the above described methyl esters l a
`and l b (6.0g, 11.5 mmol) in MeOH (50 mL), 2 N
`288
`
`NaOH (1 1.5 mL) were added. After 4 h at room tem-
`perature the reaction mixture was evaporated under
`vacuum and taken up in H20. The aqueous alkaline
`solution was washed with ether, acidified with 6 N HCl
`and extracted with chloroform. Drying and evaporation
`gave the corresponding protected tripeptide acids 2a
`and 2b as oils, pure on TLC.
`
`PhCH?CO-DSer(Bzl)-Pro-Pro-OH 2a. 5.65 g (97 o/");
`[ r]D (MeOH) -59.0'. Anal. calc. for C28H33N306: C
`66.26, H 6.55. N 8.28%. Found: C 66.30, H 6.42, N
`8.35",.
`PhCHzCO-DSer(B;l)-Pro-DPro-OH 2b. 5.7 g (987,);
`[x]D (MeOH) + 39.0". Anal. calc. for C 2 8 H d 3 0 6 : C
`66.26, H 6.55, N 8.280,;. Found: C 66.34, H 6.48, N
`8.30"".
`
`Prepuratiori oj' rripeptide active esters 4a mid 4b (Fig. 1)
`A solution of the above reported tripeptide acids 2a and
`26 (5.0 g, 9.85 mmol) in MeOH (250 mL) was hydro-
`genated in the presence of 5 2) Pd on activated charcoal
`(0.6 g). After 5 h the catalyst was filtered off and the
`solution was evaporated under reduced pressure to give
`the corresponding TLC pure deprotected tripeptide
`acids 3a or 3b (4.0g) which was used as such. To a
`solution of the tripeptide acids 3a and 3b (4.0 g,
`9.5 mmol) and p-nitrophenol (2.6 g, 19.0 rnmol) in
`CHzCI2 (160 mL), DCCl(l.95 g, 9.5 mmol) was added
`at 0 ° C with stirring. After 2 h at 0°C and 12 h at 5"C,
`the reaction mixture was filtered and the resulting so-
`lution was repeatedly washed with saturated aqueous
`NaZCO3. The residue obtained after drying and evap-
`oration was chromatographed on silicagel using EtOAc-
`:MeOH (97:3) as eluant to give the corresponding ac-
`tive esters 4a and 4b as pale yellow oils. They were used
`as such immediately after the preparation.
`
`PhCHzCO-nSer-Pro-Pro-ONp 4a. 3.1 g (60q0). Rf
`(C)=O.4; [a]D -98.0". 'H-NMR 6 = 1.8-2.5 (8H, m,
`2 x Pro P-CH2 and 2 x P r o Y - C H ~ ) , 3.5-3.9 (7H, m,
`2 x Pro 6-CH2, Ser P-CHI, OH), 3.55 (2H, s, CHzCO),
`4.6 ( l H , m, Pro r-CH), 4.7 ( l H , m Pro a-CH), 4.85
`( l H , m, Ser r-CH), 7.1 (1H. d , J = 7 . 8 Hz, NH), 7.2-
`7.4 (7H, m, ArH), 8.3 ppm (2H, two lines, ArH).
`PhCHzCO-DSer-Pro-DPro-ONp 4b. 2.75 g (54%). Rf
`(C)=0.4; [ r ] ~ +31.0". 'H-NMR 6 = 1.8-2.5 (8H, m,
`2 x Pro P-CH? and 2 x Pro y-CHz), 2.9 (lH, br s, OH),
`3.4-3.7 (5H, m, Pro 6-CH2, Pro 6-CHe, Ser P-CH?),
`3.5 (2H, s, CH2CO), 4.05 (lH, m, Pro ~ - C H A ) , 4.25
`(lH, m, Ser x-CH), 4.7 (lH, m, Pro a-CH), 5.35 (IH,
`m, Pro r-CH), 6.85 (lH, d, J = 7.0 Hz, NH), 7.2-7.35
`(7H, m, ArH), 8.25 pprn (2H, two lines, ArH).
`
`C?r.liiatiori of linear tripeptide active esters 4a arid 4b
`A solution of active ester (2.5 g, 4.6 mmol) and DBU
`(8.4 g, 5.5 mmol) in dry benzene (460 mL) was kept
`
`IPR2014-01126- Exhibit 1031 p. 7
`
`

`
`Serine-containing 1 0-membered cyclodepsipeptides
`
`20 h at room temperature. The mixture was filtered and
`the resulting solution evaporated under vacuum. The
`chloroform solution of the residue was washed with 2 N
`HCI, saturated aqueous Na2CO3 and H20, dried and
`evaporated to give a residue which was purified by
`column chromatography using CHC13:MeOH (97:3) as
`eluant. Crystallization of the collected fractions gave
`the corresponding pure cyclotridepsipeptides 5a and
`5b.
`
`PhCH2CO-oSer-Pro-Pro; 5a. 1.05 g (SOS",); m.p. 218-
`219°C (MeOH). Rf(C)=0.5; [ x ] ~ +24.0". IR (KBr)
`'H-NMR
`3390, 1750, 1640, 1625, 1495 cm- I .
`v,,,
`6 = 1.7-2.0 (4H, m, 2 x Pro y-CHz), 2.0-2.5 (2H, m,
`2 x Pro B-CH2), 3.4 (lH, m, Pro ~ - C H B ) , 3.5 (4H, m,
`CH2CO), 3.6-3.9 (4H, m, Pro ~ - C H A , Ser P-CHB, Pro
`6-CH2), 4.45 (IH, m, Pro a-CH), 3.65 (2H, m, Pro
`a-CH, Ser a-CH), 4.95 (lH, m, Ser P-CHA), 6.8 (1H
`d, J = 5.5 Hz, NH), 7.15-7.35 ppm (5H, m; ArH).
`IT-NMR 6 = 20.95 and 22.93 (Pro C;.), 30.45 and
`3 1.5 1 (Pro Cq, 43.49 (CH2CO). 47.13 and 47.69 (Pro
`C6), 49.68 (Ser C"), 58.63 and 59.80 (Pro C.), 63.99
`(Ser Cp), 127.25, 128.84, 129.26 and 134.43 (aromat-
`ics), 167.11, 170.23, 171.08 and 172.14 ppm (-CO). MS
`(FAB): m/z 400 (M + H)
`.
`
`+
`
`bond angles, torsion angles and final positional param-
`eters along with equivalent and anisotropic thermal fac-
`tors have been deposited at the Cambridge Crystallo-
`graphic Data Center.
`A4 = 487.55,
`Crystal data of5b: C ~ ~ H ~ S N ~ O ~ . E ~ O A C ,
`monoclinic, P21, from systematic ab?ences, a = 9.232(4),
`P= 97.35(3)",
`b= 11.050(6),
`C = 11.518(6) A,
`V=1165.3A3, Z = 2 , D,= 1.390g cm-',
`p(Cu
`F(OO0) = 520, sinO/A,,, = 0.61 A - I .
`Kcr) = 8.6 cm -
`During the data collection of both compounds, three
`standard reflections, measured every 100, showed only
`small random fluctuations from their means. Of the
`2228 and 2657 independent reflections collected from
`5a and 5b, respectively, 1642 and 2044 having I > 2 4 1 )
`were considered observed and used for structure solu-
`tion and refinement. Lorentz and polarization correc-
`tions were applied, but intensities were not corrected
`for extinction and absorption. The structures were
`solved by multi-solution direct methods using the pro-
`gram MULTAN 80 (19). For each structure the set
`with the highest combined figure of merit revealed al-
`most all the non-hydrogen atoms. The missing atomic
`positions were determined by structure factors and
`Fourier synthesis calculations. In the early stages of the
`structure solutions difference Fourier maps showed in
`both 5a and 5b disordered C,Y atoms as well as atoms
`of EtOAc used as crystallization solvent in 5b. All
`P/ICHzCO-DSer-ProDProl 5b. 1.2 g (65",); m.p. 161-
`atoms, except C; and 5b solvent atoms, were refined
`162"C(EtOAc). Rf(C)=0.5; [x]D -119.0'. IR(KBr)
`anisotropically. The disordered C' atoms in 5a have
`vmax 3250, 1740, 1670, 1640, 1530cm-l. 'H-NMR
`occupancies of 0.5, while those of 5b have occupancies
`6 = 1.6-2.5 (8H, m, 2 x Pro P-CIb and 2 x Pro y-CH2),
`of 0.6 and 0.4. A difference Fourier map for 5b com-
`3.2(1H,m,Pro6-CH~),3.55(2H,s,CHKO),3.7(1H,
`puted at the latest stage of the refinement showed sev-
`m, Pro ~ - C H A ) , 3.75-3.9 (2H, in, Pro b-CHz), 4.1-4.3
`eral small peaks attributable to disordered solvent mol-
`(2H, m, Ser P-CHB, Pro a-C'H), 4.7 (lH, m, Ser
`ecules. Calculations performed by using CHEM-X (20)
`P-CHA), 4.8 (lH, m, Pro a-CH), 4.95 (lH, m, Ser
`software on a videographic station, revealed that the
`cr-CH), 6.5 (lH, m , J = 7.2 Hz, NH), 7.2-7.4 ppm (5H,
`space left by the host molecules of 5b is a channel
`m, ArH). I3C-NMR 6 = 22.03 and 22.98 (Pro Cy), 32.44
`(Fig. 5 ) which allows the inclusion of EtOAc. Owing to
`and 34.56 (Pro Cp), 43.38 (CHKO), 46.27 and 49.78
`the disorder of the solvent molecule, only four non-
`(Pro Cs), 50.04 (Ser Ca), 59.49 ( 2 x Pro C.), 64.60 (Ser
`hydrogen atoms of EtOAc have been located. The hy-
`Cq; 127.40, 126.97, 129.29 and 134.32 (aromatics),
`drogen atoms in 5a as well as in 5b were generated in
`169.82, 170.69, 170.90 and 171.02 ppm (-CO). MS
`stereochemically feasible positions with isotropic ther-
`(FAB): tnjz 400 ( M + H)+ .
`mal parameters equal to that of the carrier atom and
`included and kept fixed in the last few cycles of least-
`squares calculations. The final R (and R,) are 0.057
`(and 0.079) for 5a and 0.077 (and 0.11) for 5b.
`All the calculations were carried out on a Data Gen-
`eral Eclipse MV 8000 I1 computer, with the crystallo-
`graphic software of ref. 21.
`
`X - R q dflraction
`Approximate unit cell parameters and the space groups
`of 5a and 5b were determined from oscillation and
`Weissenberg photographs. Intensity data of both com-
`pounds were recorded on an automatic four-circle dif-
`fractometer SYNTEX P21 equipped with graphite
`monochromator and Cu Kcr radiation, in 8-28 scanning
`mode to a maximum of 2 8of 140 O . The refined unit cell
`parameters were determined by least-squares refine-
`ment of the angular setting of 15 selected reflections.
`Crystal data of 5a: C21H25N305, M = 399.45, orthor-
`hombic, P212121 from systematic absences, a -5.936( l),
`b= 12.343(4), ~=27.091(11) A. Y = 1984.9A3, Z = 4 ,
`D,= 1.337g~m-~~,p(CuKcr)=
`X . l cm-l,F(000)=848,
`sin8/Amax = 0.61 A - A complete list of bond lengths,
`
`ACKNOWLEDGMENTS
`
`This work has been supported by the Minister0 dell'UniversIti e della
`Ricerca Scientifica e Tecnologica (Italy). We thank Prof. G. Lucente
`for helpful discussions and Mr. B. Trabassi for the drawings.
`
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`
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`
`289
`
`IPR2014-01126- Exhibit 1031 p. 8
`
`

`
`Cerrini et al.
`
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