1. Defrag

for example with MyDefrag - it has a option to put empty space at the end

2. zero empty space

use sdelete from https://technet.microsoft.com/en-us/sysinternals/sdelete.aspx

sdelete -z c:

3. Compact VDI

VBoxManage modifyvdi --compact /path/to/disc.vdi   # use uuid of disc, if this doesn't work