Conjugation Chemistry and the Preparation of Polymer-Drug Conjugates
PHPMA 1 derived from PM OSu 36 conventional PHPMA
dextran p oly-L -lysine 10 .001 .01 I p olym er! /m g -m l *
F ig u r e 4 .5 . C y to to x ic ity o f P H P M A 1 prepared via P M O S u 3 6 and co n v en tio n a l 1 u sin g B 1 6 F 1 0 c e lls o v e r 7 2 h (m ean ± S D ).
2 .3 C o m p e titiv e h y d r o ly s is
T o in v e stig a te the p o ssib ility o f P M O S u 3 6 u n d ergoin g c o m p e titiv e h y d ro ly sis during a m in o ly sis resu ltin g in the form ation o f undesired m eth acrylic acid repeat units on the p olym er, several e x p erim en ts w ere perform ed. F irstly, a sa m p le o f 3 6 w a s
C h a p te r 4 : C o n ju g a tio n C h e m istr y
o
r ONa+ 62 \h y d r o ly se d u sin g a q u eo u s N a O H to the so d iu m salt o f P M A A 62 (s e e S c h e m e 5 .2 and se c tio n 2.1 for sy n th e sis, C hapter 5 ). In the IR, this c o m p o u n d g a v e a C O2 stretch band at - 1 5 5 9 c m '. T h e p ro to n a ted
form o f this p o ly m e r w a s ob tain ed b y the ad d ition o f H C l and the so lid product g a v e a carb on yl stretch at the higher freq u en cy o f 1703 c m '. T h is su g g e ste d that the p resen ce
o f proton ated ca r b o x y lic acid repeat units m ay be r e sp o n sib le for the sh o u ld er band o b s e r v e d at arou n d 1 7 0 0 - 1 7 1 0 c m ' se e n to v a r y in g d e g r e e s in th e c o n ju g a tio n r e a c tio n s o f 36 w ith I-a m in o -2 -p r o p a n o l 10 (a s d e sc r ib e d in s e c tio n 2.1) d u e to c o m p e titiv e h y d ro ly sis.
T o further in v e stig a te c o m p e titiv e h y d r o ly sis, a sa m p le o f 36 in D M S O w a s a llo w e d to react w ith a q u e o u s 10 to prom ote h y d r o ly sis (e x p e rim e n ta l se c tio n 4.1, C h ap ter 7 ). IR a n a ly sis o f th e p r o d u ct s h o w e d a p r o m in e n t b a n d at 1 7 1 0 c m ' p ro v id in g e v id e n c e for c o m p e titiv e h y d r o ly s is (sp ectru m A , F igu re 4 .6 ). T h is sa m p le w a s then d is s o lv e d in d ilu te N aO H . A n IR sp ectru m o f the so lid iso la te d fro m the N a O H so lu tio n sh o w e d the near d isa p p ea ra n ce o f th e band at 1 7 1 0 cm ' and an in c r e a se at 1 5 5 0 cm ' (sp ec tru m B , F ig u r e 4 .6 ) . T h is is c o n siste n t w ith the p r e se n c e o f M A A N a salt repeat units in the p o ly m e r , w h ic h w o u ld b e e x p e c te d i f c a r b o x y lic a c id g rou p s in the p o ly m e r w ere treated w ith N a O H . T h e sa m p le w a s th en d is s o lv e d in H C l to r e -p r o to n a te the su sp e c te d ca rb o x y la te grou p s. A fter iso la tio n o f the p o ly m er , the band at - 1 7 1 0 cm ' had returned in the IR sp e ctru m (sp e ctru m C, F igu re 4 .6 ) further su p p o rtin g the h y p o th e s is o f c o m p e titiv e h y d ro ly sis. o 10
t
A 1750 1700 1650 1600 1550 w avenum ber/cm * Figure 4.6. (A ) FT -IR (carbonyl region ) o f P H P M A derived from P M O Su; (B ) after treatm ent w ith base; (C ) after treatm ent w ith acid.C h a p te r 4: C o n ju g a tio n C h e m is tr y
A n a ly sis o f the sam p le by ’H N M R a lso in d icated h y d ro ly sis, sin c e the b ack b on e m eth y len e and m ethyl protons had larger in tegrals than w o u ld be e x p ec ted for ju st H P M A h o m o p o ly m e r 1. T h e ratio o f H P M A to M A A repeat units w a s c a lcu la ted to be ap p roxim ately 1.5:1 r e sp e c tiv e ly from the N M R integral v a lu es, i.e, 40% h y d ro ly sis had occurred. T h e ex ten t o f h y d r o ly sis in a sa m p le o f P H P M A 1 prepared from P M O S u 3 6 in se c tio n 2 .1 , i.e ., w ith o u t the addition o f w ater to the reaction m ixture, w a s calcu lated to be a p p ro x im a tely 4% .
T o further a sse ss the h y d ro ly tic sta b ility o f 3 6 in so lu tio n , an anhydrous D M S O so lu tio n o f 3 6 w a s heated w ith trieth ylam in e (0 .5 e q u iv .) at 6 0 °C. A fter 1 h, the IR spectra o f the starting and heated sa m p le s w ere id en tica l, in d icatin g no h y d r o ly sis had occurred. H o w ev er , after 2 h a 13% d ecrea se in peak h eig h t m easured at 1735 cm ‘ w a s o b serv ed . A fter 16 h at 6 0 °C the d ecrea se b ecam e 22% in d icatin g that the p o ly m e r w ill undergo h y d r o ly sis w ith h eatin g in the p resen ce o f base and trace w ater from the so lv e n t. It w a s a lso o b serv ed that com pared to fresh ly prepared 3 6 , sa m p le s o f 3 6 that had been stored under am b ien t co n d itio n s for several m onths had a slig h t sh ou ld er band in the IR at around 1 7 1 0 cm ' that in d icated h y d r o ly sis had occurred (carb on yl band o f ca rb o x y lic acid grou p s. Figure 4 .7 ).
A A e v id e n c e o f h y d r o ly sis \ o n storage I860 1840 1820 1800 1780 1760 1740 1720 1700 1680 1660 wavenumber/cm' '
F ig u r e 4 .7 . FT-IR sp ectr o sc o p y co m p a riso n o f fresh (b lu e sp ectru m ) and stored (red sp ectru m ) sa m p les o f P M O S u 3 6 .
Chapter 4: Conjugation Chemistry
With and NMR there was no distinction between the stored and freshly
prepared 36, as there was no signal at approximately Ô 12.28 (broad) relating to
COOH (value obtained from analysis of hydrolysed 36 in DMSO-Jg) in ’H NMR
analysis and no evidence of the carbonyl carbon of the acid (approx. 5 180) in analysis. However, elemental combustion analysis (Table 4.1) revealed discrepancies between theoretical and experimental values; not only in the stored samples of 36 but also the fresh samples, suggesting the polymer samples tested may have undergone some very slight hydrolysis during the polymerisation (at 70 to 100 °C) and/or isolation and purification.
% C % H % N
T h e o r y b a s e d o n C g H ^ N O ^ r e p e a t u n i t 52.46 4.95 7.65
Found
Fresh sample* 50.82 5.18 7.50
Stored sample 47.52 5.38 6.64
Table 4.1. Elemental combustion analysis of PMOSu 36 samples; (*) average of five different samples.
Competitive hydrolysis cannot occur in the absence of water from the reaction. However, the hygroscopic nature of DMSO and DMF makes water exclusion difficult to achieve in practice. An examination of different solvents (perhaps mixed solvent) systems that are less hygroscopic may be a solution to this. Alternatively, a longer- term solution may be to prepare copolymer precursors to confer solubility in other less polar organic solvents. As concluded earlier, however, the use of a large excess of nucleophile in the preparation of homopolymers (e.g., 100 equiv. excess) is one way to minimisation/eliminate the shoulder band at -1710 cm^ in the solid product.
C h a p te r 4: C o n ju g a tio n C h e m istr y
2.4 C o m p e t i t i v e im id e f o r m a t i o n
C o m p e titiv e c y c lic im id e and a n h y d rid e fo r m a tio n ( S c h e m e 4 .2 ) are w e ll k n o w n s id e r e a c tio n s o f r e a c tiv e p o ly m e r s (A rsh a rd y , 1 9 9 4 ; S tr o h r ie g l, 1 9 9 3 ). A m ec h a n ism o f anhydride form ation for 3 6 in the preparation o f p o ly (m eth a c ry la m id e s) is d u e to th e h y d r o ly s is o f an a c tiv e e ste r g ro u p f o llo w e d b y r e a c tio n w ith the n eig h b o u rin g a c tiv e ester (S c h e m e 4 .2 , A ).
pHs <pi-b pHa pHa
p CH2 p CH2---
o - ç p
pH3 pHî pHî pHa
'CH2 p CH2 p CH2-
NIH p ^ p
Scheme 4 .2 . A n h yd rid e (A ) and im id e (B ) form ation b e tw e e n n eig h b o u rin g groups.
S in c e h y d r o ly sis can th e o r e tic a lly b e p rev en ted b y the v ig o r o u s e x c lu s io n o f w a ter or s u b s ta n tia lly r e d u c e d or e lim in a tio n b y th e u s e o f a la r g e e x c e s s o f n u c l e o p h i l i c r e a c t a n t , a n h y d r id e f o r m a t i o n c a n t h e r e f o r e a l s o b e elim in a te d /m in im ise d . If c y c lic anhydride grou p s w ere p resen t it can b e e x p e c te d that carb on yl bands in the reg io n s 1 8 7 0 to 1845 cm ' (sy m m etr ic stretch) and 1 8 0 0 to 1775 c m ' (a n ti-sy m m etr ic stretch) w o u ld be o b se r v e d in the IR (V ie e t a l , 1 9 9 1 ). T h is is not c o n siste n t w ith the bands seen at 1772 cm ' and around 1 7 1 0 c m ' (sh o u ld er) in the reaction s to prepare H P M A h o m p o ly m er 1 in se c tio n 2 .1 (s e e spectrum C , F igure 4 .2 ).
C h a p te r 4: C o n ju g a tio n C h e m istr y
H o w e v e r , c y c lic im id e b an d s are c o n s is te n t w ith th e s e w a v e n u m b e r s s in c e th ey ty p ic a lly o c cu r at a lo w e r freq u en cy , 1 8 0 0 to 1735 cm * (sy m m e tr ic stretch) and 1 7 5 0 to 1 6 8 0 c m * (a n ti-sy m m e tr ic stretch ) (V ie e t a l , 1 9 9 1 ). T h e sa m p le o f 1 prepared from 36 w h ere the band at 1 7 7 2 cm * w as m o st p rom in en t a lso ex h ib ite d a large sig n a l at Ô 2 .3 in a * H N M R sp ectru m (la b e lle d h in F ig u re 4 .8 ) w h ic h w a s n ot p resen t in c o n v e n tio n a l P H P M A 1 sp ectru m . T h is s u g g e s ts that th is N M R sig n a l is d u e to a structural m o ie ty a lso r esp o n sib le for the ad d ition al IR band at 1 7 7 2 c m *.
CH2 r i C O NH CH, HO OH CH3 1 00. CO b' b ' yH 3 C H , Chfe— ( j î - C H j - N" CH, H C - OH CH3 b + g I
&
500 PPM /. 00 3 00Figure 4.8. *H N M R sp ectru m o f P H P M A 1 d er iv ed from P M O S u 36 that from IR
a n a ly s is (sp ectr u m C , F ig u re 4 .2 ) m ay p o s s e s s im id e im p u rity in the b ack b o n e sim ilar to the sim p lifie d structure sh o w n .
In a d d itio n to the im id e fo r m a tio n fo r m ed b y the re a c tio n o f im m e d ia te ly a d ja cen t a m id e and a c tiv e e ste r rep ea t u n its ( S c h e m e 4 .2 , B ) th ere is a ls o the p o ssib ility o f c r o ss-lin k in g b e tw een n o n -n eig h b o u rin g fu n ctio n a l g rou p s in a p o ly m e r or b e tw e e n separate p o ly m e r ch a in s lea d in g to c r o ss-lin k in g and a b roa d en in g o f the M W D . T o probe th is, an e x p e r im e n t w a s p erfo rm ed u sin g a lo w M W a c tiv e ester
Chapter 4: Conjugation Chemistry
m o d e l c o m p o u n d , A ^ -p rop ion yioxysu ccin im id e 63 w ith n -p r o p y la m in e 64 (S c h e m e 4 .3 , s e e a lso ex p erim en tal sectio n 4.3, C hapter 7).
O
50 °C
° ^ DM SO, 50 °C H
63 64
Scheme 4.3. In v e stig a tio n o f am id e and im id e form ation u sin g lo w MW su c c in im id y l
ester m od el 63.
U n d er sim ila r c o n d itio n s (i.e ., c o n c en tra tio n and tem p eratu re) e m p lo y e d for c o n ju g a tio n r e a c tio n s in v o lv in g P M O S u 36, n o e v id e n c e o f th e im id e 65 w a s o b tain ed , in d ica tin g that c o m p e titiv e im id e fo rm ation for 36 m ay be s o le ly a p o ly m e r e ffe c t d ue to the c lo s e p ro x im ity o f ad jacen t n e ig h b o u rin g fu n c tio n a l g ro u p s and the lik e ly fo rm a tio n o f e n e r g e tic a lly fa v o u r e d s ix -m e m b e r e d r in g s (S tr o h r ie g l, 1 9 9 3 ). H o w e v e r , th is re a ctio n m ay not b e r ep r e se n ta tiv e o f a p o ly m e r r e a c tio n w h ic h is